Pyrazolylbenzothiazole derivatives and their use as therapeutic agents

ABSTRACT

Topical formulations and method of using the same are provided. The topical formulation comprises a pyrazolylbenzothiazole derivative of the following formula (1) 
                         
wherein X, R 1 , R 2 , R 3  and R 4  are described herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. application Ser.No. 12/910,744, filed Oct. 22, 2010 (allowed), which is a continuationapplication of U.S. application Ser. No. 10/521,948, filed Jan. 23, 2006(U.S. Pat. No. 7,847,101), which is a §371 national phase conversion ofInternational Patent Application No. PCT/CA2003/001078, filed Jul. 23,2003, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 60/398,504, filed Jul. 24, 2002. Allof the above applications are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention is directed to pyrazolylbenzothiazole derivatives,pharmaceutical compositions containing the derivatives and methods ofusing the derivatives as therapeutic agents.

BACKGROUND OF THE INVENTION

It has become increasingly clear in recent years that cell death is asimportant to the health of a multicellular organism as cell division;where proliferation exists, so must a means of regulating its cellularprogeny. By repeated cell division and differentiation throughoutdevelopment or tissue repair, surplus or even harmful cells aregenerated, and they must be removed or killed. In adults, senescentcells are removed and replaced by newly generated cells to maintainhomeostasis.

The delicate interplay between growth and cell death in an organism ismirrored in the complex molecular balance that determines whether anindividual cell undergoes division; arrests in the cell cycle; orcommits to programmed cell death. Signal transduction is the termdescribing the process of conversion of extracellular signals, such ashormones, growth factors, neurotransmitters, cytokines, and others, to aspecific intracellular response such as gene expression, cell division,or apoptosis. This process begins at the cell membrane where an externalstimulus initiates a cascade of enzymatic reactions inside the cell thattypically include phosphorylation of proteins as mediators of downstreamprocesses which most often end in an event in the cell nucleus. Thechecks and balances of these signal transduction pathways can be thoughtof as overlapping networks of interacting molecules that control “go-nogo” control points. Since almost all known diseases exhibitdysfunctional aspects in these networks, there has been a great deal ofenthusiasm for research that provides targets and therapeutic agentsbased on signal transduction components linked to disease.

Dysregulation of cell proliferation, or a lack of appropriate celldeath, has wide ranging clinical implications. A number of diseasesassociated with such dysregulation involve hyperproliferation,inflammation, tissue remodelling and repair. Familiar indications inthis category include cancers, restenosis, neointimal hyperplasia,angiogenesis, endometriosis, lymphoproliferative disorders,graft-rejection, polyposis, loss of neural function in the case oftissue remodelling, and the like. Such cells may lose the normalregulatory control of cell division, and may also fail to undergoappropriate cell death.

In one example, epithelial cells, endothelial cells, muscle cells, andothers undergo apoptosis when they lose contact with extracellularmatrix, or bind through an inappropriate integrin. This phenomenon,which has been termed “anoikis” (the Greek word for “homelessness”),prevents shed epithelial cells from colonizing elsewhere, thusprotecting against neoplasia, endometriosis, and the like. It is also animportant mechanism in the initial cavitation step of embryonicdevelopment, in mammary gland involution, and has been exploited toprevent tumor angiogenesis. Epithelial cells may become resistant toanoikis through overactivation of integrin signaling. Anoikis resistancecan also arise from the loss of apoptotic signaling, for example, byoverexpression of Bcl-2 or inhibition of caspase activity.

An aspect of hyperproliferation that is often linked to tumor growth isangiogenesis. The growth of new blood vessels is essential for the laterstages of solid tumor growth. Angiogenesis is caused by the migrationand proliferation of the endothelial cells that form blood vessels.

In another example, a major group of systemic autoimmune diseases isassociated with abnormal lymphoproliferation, as a result of defects inthe termination of lymphocyte activation and growth. Often such diseasesare associated with inflammation, for example with rheumatoid arthritis,insulin dependent diabetes mellitus, multiple sclerosis, systemic lupuserythematosus, and the like. Recent progress has been made inunderstanding the causes and consequences of these abnormalities. At themolecular level, multiple defects may occur, which result in a failureto set up functional apoptotic machinery.

The development of compounds that inhibit hyperproliferative diseases,particularly where undesirable cells are selectively targeted, is ofgreat medical and commercial interest.

RELATED LITERATURE

The regulation of integrin linked kinase by phosphatidylinositol (3,4,5)trisphosphate is described by Delcommenne et al. (1998) Proc Natl AcadSci 95:11211-6. Activated nitriles in heterocyclic synthesis arediscussed in Kandeel et al. (1985) J. Chem. Soc. Perkin. Trans 1499.

SUMMARY OF THE INVENTION

The invention is directed to pharmaceutical compositions comprising apharmaceutically acceptable excipient and a compound of formula (1):

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: R¹, R² and R³ at eachoccurrence is independently selected from amino, aminosulfinyl,aminosulfonyl, aryl, azido, halogen, heteroalkyl, heteroaryl,hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso, phosphate,phosphinate, phosphonate, phosphonium, phosphorothioate, phosphoryl,sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate, sulfonicacid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R⁴ is selectedfrom hydrogen, heteroalkyl, heteroaryl, and hydrocarbyl; X is selectedfrom S, O and NR⁹, and R⁹ is selected from hydrogen, heteroalkyl,heteroaryl, and hydrocarbyl.

These compounds of the invention will be referred to herein aspyrazolylbenzothiazole compounds or derivatives or analogs, where theseterms are used interchangeably. Technically, a benothiazole compound hasX equal to S. However, when X is O or NR⁹, then the compounds may beconsidered pyrazolylbenzothiazole analogs.

In another aspect, this invention is directed to certain compounds offormula (1). For example, compounds of formula (1) as a single tautomer,a mixture of tautomers, a single stereoisomer, a mixture ofstereoisomers, or a racemic mixture; or a pharmaceutically acceptablesalt or solvate thereof; wherein: X is selected from S, O and NR⁹, R⁹ isselected from hydrogen, heteroalkyl, heteroaryl, and hydrocarbyl; R¹ andR² at each occurrence is independently selected from amino,aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R³ isheterocycle; and R⁴ is selected from hydrogen, heteroalkyl, heteroaryl,and hydrocarbyl.

As another example, the invention provides compounds of formula (1) as asingle tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ each occurrence is independently selected from amino,aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R² isamino; R³ is selected from hydrocarbyl, —O-hydrocarbyl and—S-hydrocarbyl; and R⁴ is selected from hydrogen, heteroalkyl,heteroaryl, and hydrocarbyl.

As another example, the present invention provides compounds of formula(1) as a single tautomer, a mixture of tautomers, a single stereoisomer,a mixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ and R² at each occurrence is independently selected fromamino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R³ ishydrogen; and R⁴ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl.

As another example, the present invention provides compounds of formula(1) as a single tautomer, a mixture of tautomers, a single stereoisomer,a mixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ and R² at each occurrence is independently selected fromamino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R³ ishydrocarbyl; and R⁴ is selected from hydrogen, heteroalkyl, heteroaryl,and hydrocarbyl.

As another example, the present invention provides compounds of formula(1) as a single tautomer, a mixture of tautomers, a single stereoisomer,a mixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹, R² and R³ at each occurrence is independently selectedfrom amino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen,heteroalkyl, heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl,nitro, nitroso, phosphate, phosphinate, phosphonate, phosphonium,phosphorothioate, phosphoryl, sulfamoyl, sulfate, sulfinic acid,sulfonamido, sulfonate, sulfonic acid, sulfonyl, sulfoxido, thiol,thioureido, and ureido, with the proviso that R¹ is not hydrogen in atleast one occurrence of R¹; and R⁴ is selected from hydrogen,heteroalkyl, heteroaryl, and hydrocarbyl.

As another example, the present invention provides compounds of formula(1) as a single tautomer, a mixture of tautomers, a single stereoisomer,a mixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ and R² at each occurrence is independently selected fromamino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R³ ishalogen-substituted hydrocarbyl; and R⁴ is selected from hydrogen,heteroalkyl, heteroaryl, and hydrocarbyl.

In another aspect, the present invention provides compounds of formula(2)

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ at each occurrence is independently selected from amino,aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R⁴ isselected from hydrogen, heteroalkyl, heteroaryl, and hydrocarbyl; andR⁵, R⁶, R⁷ and R⁸ at each occurrence is independently selected fromheteroalkyl, heteroaryl, hydrocarbyl and hydrogen, with the proviso thatR⁷ and R⁸ may join together to form a heterocyclic ring including thenitrogen to which they are both bonded.

In related aspects, the present invention provides pharmaceuticalcompositions including compounds as set forth in an aspect as describedabove, in combination with a pharmaceutically acceptable excipient.

In another aspect, this invention is directed to the use of, and methodsof using, the compounds of formula (1) as described above in thetreatment of disorders associated with hyperproliferation and tissueremodelling or repair, inflammation, cell migration and invasion, andrenal disease. The compounds are also useful in the inhibition ofspecific protein kinases, such as integrin-linked kinase.

In another aspect, the present invention provides compounds of formula(3):

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: R² and R³ at eachoccurrence is independently selected from amino, aminosulfinyl,aminosulfonyl, aryl, azido, halogen, heteroalkyl, heteroaryl,hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso, phosphate,phosphinate, phosphonate, phosphonium, phosphorothioate, phosphoryl,sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate, sulfonicacid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R⁴ is selectedfrom hydrogen, heteroalkyl, heteroaryl, and hydrocarbyl; X is selectedfrom S, O and NR⁹, and R⁹ is selected from hydrogen, heteroalkyl,heteroaryl, and hydrocarbyl; Y is a 6 membered heterocycle having 1 or 2nitrogen atoms and which is optionally further substituted by one ormore groups selected from amino, aminosulfinyl, aminosulfonyl, aryl,azido, halogen, heteroalkyl, heteroaryl, hydrazinyl, hydrocarbyl,hydrogen, hydroxyl, nitro, nitroso, phosphate, phosphinate, phosphonate,phosphonium, phosphorothioate, phosphoryl, sulfamoyl, sulfate, sulfinicacid, sulfonamido, sulfonate, sulfonic acid, sulfonyl, sulfoxido, thiol,thioureido, and ureido.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention provides novel compounds, compositions and methodsas set forth within this specification. In general, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs, unless clearly indicated otherwise. For clarification, listedbelow are definitions for certain terms used herein to describe thepresent invention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise clearly indicated.

DEFINITION OF TERMS

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. For example, “acompound” refers to one or more of such compounds, while “the enzyme”includes a particular enzyme as well as other family members andequivalents thereof as known to those skilled in the art. As used in thespecification and appended claims, unless specified to the contrary, thefollowing terms have the meaning indicated:

“Alkyl” refers to a straight or branched monovalent hydrocarbon chainradical consisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to eight carbon atoms, and which isattached to the rest of the molecule by a single bond, e.g., methyl,ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), and the like. Unless stated otherwisespecifically in the specification, it is understood that for radicals,as defined below, that contain a substituted alkyl or alkenyl group thatthe substitution can occur on any carbon of the alkyl group.

“Alkylene chain” refers to a straight or branched divalent hydrocarbonchain consisting solely of carbon and hydrogen, containing nounsaturation and having from one to eight carbon atoms, e.g., methylene,ethylene, propylene, n-butylene, and the like.

“Alkenyl” refers to a straight or branched monovalent hydrocarbon chainradical consisting solely of carbon and hydrogen atoms, containing atleast one double bond, having from two to eight carbon atoms, and whichis attached to the rest of the molecule by a single bond, e.g., ethenyl,prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above, e.g., methoxy, ethoxy, n-propoxy,1-methylethoxy (iso-propoxy), n-butoxy, n-pentoxy, 1,1-dimethylethoxy(t-butoxy), and the like.

“Aryl” refers to a phenyl or naphthyl radical. Unless stated otherwisespecifically in the specification, the term “aryl” or the prefix “ar-”(such as in “aralkyl”) is meant to include aryl radicals optionallysubstituted by one or more substituents selected from the groupconsisting of alkyl, alkoxy, hydroxy, halo, haloalkyl, haloalkoxy, aminoand carboxy as defined herein.

“Aralkyl” refers to a radical of the formula —R_(a)R_(b) where R_(a) isan alkyl radical as defined above and R_(b) is one or more aryl radicalsas defined above, e.g., benzyl, diphenylmethyl, and the like. The arylradical may be optionally substituted as described above.

“Aralkenyl” refers to a radical of the formula —R_(e)—R_(b) where R_(b)is an aryl radical as defined above and R_(e) is an alkenyl radical asdefined above, e.g., 2-phenylethenyl, and the like.

“Carboxy” refers to the —C(O)OH radical.

“Cycloalkyl” refers to a stable monovalent monocyclic or bicyclichydrocarbon radical consisting solely of carbon and hydrogen atoms,having from three to ten carbon atoms, and which is saturated andattached to the rest of the molecule by a single bond, e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decalinyl and thelike. Unless otherwise stated specifically in the specification, theterm “cycloalkyl” is meant to include cycloalkyl radicals which areoptionally substituted by one or more substituents independentlyselected from the group consisting of alkyl, alkoxy, halo, haloalkyl,haloalkoxy, hydroxy, amino, and carboxy.

“Halo” refers to bromo, chloro, iodo or fluoro.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl,1-bromomethyl-2-bromoethyl, and the like.

“Haloalkoxy” refers to a radical of the formula —OR_(c) where R_(c) isan haloalkyl radical as defined above, e.g., trifluoromethoxy,difluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy,1-fluoromethyl-2-fluoroethoxy, 3-bromo-2-fluoropropoxy,1-bromomethyl-2-bromoethoxy, and the like.

“Heterocyclyl” refers to a stable 3- to 15-membered ring radical whichconsists of carbon atoms and from one to five heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur. For purposes ofthis invention, the heterocyclyl radical may be a monocyclic, bicyclicor tricyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclyl radical may be aromatic or partiallyor fully saturated. The heterocyclyl radical may not be attached to therest of the molecule at any heteroatom atom. Examples of suchheterocyclyl radicals include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl, carbazolyl,cinnolinyl, decahydroisoquinolyl, dioxolanyl, furanyl, furanonyl,isothiazolyl, imidazolyl, imidazolinyl, imidazolidinyl,isothiazolidinyl, indolyl, isoindolyl, indolinyl, isoindolinyl,indolizinyl, isoxazolyl, isoxazolidinyl, morpholinyl, naphthyridinyl,oxadiazolyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, oxazolyl,oxazolidinyl, oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, pyridinyl,pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl,quinolinyl, quinuclidinyl, isoquinolinyl, thiazolyl, thiazolidinyl,thiadiazolyl, triazolyl, tetrazolyl, tetrahydrofuryl, triazinyl,tetrahydropyranyl, thienyl, thiamorpholinyl, thiamorpholinyl sulfoxide,and thiamorpholinyl sulfone. Unless stated otherwise specifically in thespecification, the term “heterocyclyl” is meant to include heterocyclylradicals as defined above which are optionally substituted by one ormore substituents selected from the group consisting of hydroxy, halo,alkyl, alkoxy, haloalkyl, haloalkoxy, nitro, cyano, amino, and carboxy.Preferred heterocyclyl radicals for R⁵ are those radicals selected fromthe group consisting of furanyl, isooxazolyl, pyridinyl, thienyl,pyrrolyl, quinolinyl, benzothienyl, benzodioxolyl, benzooxadiazolyl,pyrazole, thiadiazolyl, and quinoxalinyl;

“N-heterocyclyl” refers to a heterocyclyl radical as defined abovewherein the one to five heteroatoms contained therein are selected onlyfrom nitrogen. Preferred N-heterocyclyl radicals for R² are thoseradicals selected from the group consisting of pyridinyl, thiazolyl,tetrazolyl, pyrazolyl, isoquinolinyl, quinolinyl, and phthalazinyl;

“Heterocyclylalkyl” refers to a radical of the formula —R_(a)R_(d) whereR_(a) is an alkyl radical as defined above and R_(d) is a heterocyclylradical as defined above, and if the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkyl radical at the nitrogen atom. A preferred heterocyclylalkylradical for R³ is morpholinylalkyl; preferred heterocyclylalkyl radicalsfor R⁵ are those radicals selected from the group consisting ofisoindoledionylalkyl, morpholinylalkyl, and triazolylalkyl.

“Heterocyclylcarbonyl” refers to a radical of the formula —C(O)—R_(d)where R_(d) is a heterocyclyl radical as defined above, and if theheterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl maybe attached to the carbonyl at the nitrogen atom. A preferredhetereocyclylcarbonyl radical for R³ is pyridinylcarbonyl.

“Hydrocarbyl”, sometimes abbreviated as “Hy”, refers to a radicalcomposed solely of carbon and hydrogen. The hydrocarbyl group may besaturated or unsaturated, and may independently have the carbonsarranged in a linear, branched or cyclic fashion. In various optionalembodiments of the invention, the hydrocarbyl moiety has 1-100, or 1-90,or 1-80, or 1-70, or 1-60, or 1-50, or 1-45, or 1-40, or 1-35, or 1-30,or 1-29, or 1-28, or 1-27, or 1-26, or 1-25, or 1-24, or 1-23, or 1-22,or 1-21, or 1-20, or 1-19, or 1-18, or 1-17, or 1-16, or 1-15, or 1-14,or 1-13, or 1-12, or 1-11, or 1-10, or 1-9, or 1-8, or 1-7, or 1-6, or1-5, or 2-100, or 2-90, or 2-80, or 2-70, or 2-60, or 2-50, or 2-45, or2-40, or 2-35, or 2-30, or 2-29, or 2-28, or 2-27, or 2-26, or 2-25, or2-24, or 2-23, or 2-22, or 2-21, or 2-20, or 2-19, or 2-18, or 2-17, or2-16, or 2-15, or 2-14, or 2-13, or 2-12, or 2-11, or 2-10, or 2-9, or2-8, or 2-7, or 2-6, or 2-5, or 3-100, or 3-90, or 3-80, or 3-70, or3-60, or 3-50, or 3-45, or 3-40, or 3-35, or 3-30, or 3-29, or 3-28, or3-27, or 3-26, or 3-25, or 3-24, or 3-23, or 3-22, or 3-21, or 3-20, or3-19, or 3-18, or 3-17, or 3-16, or 3-15, or 3-14, or 3-13, or 3-12, or3-11, or 3-10, or 3-9, or 3-8, or 3-7, or 3-6, or 3-5 carbons.Independently, the hydrocarbyl moiety may be described as an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkylene, or aryl moiety, wherealkyl, alkenyl and alkynyl is optionally substituted with one or moreHy¹ groups selected from cycloalkyl, cycloalkylene and aryl, where eachHy¹ group is optionally substituted with one or more Hy² groups selectedfrom alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylene, and aryl; andcycloalkyl, cycloalkylene and aryl is optionally substituted with one ormore Hy² groups, provided that when Hy² is selected from alkyl, alkenylor alkynyl, then Hy² may be substituted with one or more Hy³ groupsselected from cycloalkyl, cycloalkylene and aryl, where each Hy³ groupis optionally substituted with one or more Hy⁴ groups selected fromalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylene, and aryl, and whenHy² is selected from cycloalkyl, cycloalkylene and aryl then Hy² isoptionally substituted with one or more Hy⁴ groups, and further providedthat aryl includes an aryl ring fused to a non-aromatic hydrocarbocyclicring. Halogen-substituted hydrocarbyl refers to a hydrocarbyl groupwherein one or more of the hydrogens has been replaced with an equalnumber of halogens.

“Mammal” includes humans and domesticated animals, such as cats, dogs,swine, cattle, sheep, goats, horses, rabbits, and the like.

As used herein, “methods known to one of ordinary skill in the art” maybe identified though various reference books and databases. Suitablereference books and treatise that detail the synthesis of reactantsuseful in the preparation of compounds of the present invention, orprovide references to articles that describe the preparation, includefor example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., NewYork; S. R. Sandler et al., “Organic Functional Group Preparations,” 2ndEd., Academic Press, New York, 1983; H. O. House, “Modern SyntheticReactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure”, 4th Ed., Wiley-Interscience, New York, 1992. Specificand analogous reactants may also be identified through the indices ofknown chemicals prepared by the Chemical Abstract Service of theAmerican Chemical Society, which are available in most public anduniversity libraries, as well as through on-line databases (the AmericanChemical Society, Washington, D.C., www.acs.org may be contacted formore details). Chemicals that are known but not commercially availablein catalogs may be prepared by custom chemical synthesis houses, wheremany of the standard chemical supply houses (e.g., those listed above)provide custom synthesis services.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid and the like, andorganic acids such as acetic acid, trifluoroacetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltsthat retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline and caffeine.

“Pharmaceutically acceptable excipient” as used herein is intended toinclude without limitation any adjuvant, carrier, excipient, glidant,sweetening agent, diluent, preservative, dye/colorant, flavor enhancer,surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, emulsifier, or stabilizer which hasbeen approved by the United States Food and Drug Administration as beingacceptable for use in humans or domestic animals.

“Prodrugs” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the invention. Prodrugs are typically rapidly transformed invivo to yield the parent compound of the invention, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam).

A discussion of prodrugs is provided in Higuchi, T., et al., “Pro-drugsas Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14, and inBioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated in full by reference herein.

The term “prodrug” is also meant to include any covalently bondedcarriers that release the active compound of the invention in vivo whensuch prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of the invention. Prodrugs include compounds of theinvention wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the compound of the invention isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol and amine functional groups in the compounds ofthe invention and the like.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

Tautomers refer to various forms of a compound that differ only by theshifting of one or more double bonds and the concomitant shifting ofhydrogen atoms. For example, when R⁴ is hydrogen in the formula (1),then the two double bonds may shift to provide two tautomeric forms, asshown in formulae (1a) and (1b).

Another example of tautomerism arises when a substituent on the pyrazolering is adjacent to a double bond and can have hydrogen substitution. Aspecific example is shown in formulae (1c) and (1d).

“Therapeutically effective amount” refers to that amount of a compoundof the invention, which, when administered to a mammal, particularly ahuman, in need thereof, is sufficient to effect treatment, as definedbelow, for hyperproliferative disorders. The amount of a compound of theinvention which constitutes a “therapeutically effective amount” willvary depending on the compound, the hyperproliferative disorder and itsseverity, and the age of the mammal to be treated, but can be determinedroutinely by one of ordinary skill in the art having regard to his ownknowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of ahyperproliferative disorder in a mammal, preferably a human, andincludes:

(i) preventing the disorder from occurring in a human, in particular,when such mammal is predisposed to the disorder but has not yet beendiagnosed as having it;

(ii) inhibiting the disorder, i.e., arresting its development; or

(iii) relieving the disorder, i.e., causing regression of the disorder.

The compounds of the invention, or their pharmaceutically acceptablesalts, may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as, their racemic and optically pureforms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques. When the compounds described herein containolefinic double bonds or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds includeboth E and Z geometric isomers.

Unless otherwise indicated by the nomenclature, compound names areintended to include any single tautomer, single stereoisomer,enantiomer, racemate or mixtures thereof.

PREFERRED EMBODIMENTS

As mentioned previously, in one aspect the present invention provides apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a compound of formula (1):

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹, R² and R³ at each occurrence is independently selectedfrom amino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen,heteroalkyl, heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl,nitro, nitroso, phosphate, phosphinate, phosphonate, phosphonium,phosphorothioate, phosphoryl, sulfamoyl, sulfate, sulfinic acid,sulfonamido, sulfonate, sulfonic acid, sulfonyl, sulfoxido, thiol,thioureido, and ureido; and R⁴ is selected from hydrogen, heteroalkyl,heteroaryl, and hydrocarbyl. In various optional embodiments of thepresent invention, the compositions containing a compound of formula (1)may be described as containing a compound of formula (1) wherein one ormore of the following criteria are used to describe the compounds offormula (1), where any two or more of these criteria may be combined indescribing a group of compounds of formula (1) that may be present inthe pharmaceutical composition of the invention: heteroalkyl is one ormore of the following: aminohydrocarboyl (i.e., —NH—C(═O)—Hy), amido(i.e., —C(═O)—NH₂), carboxylic acid (i.e., —COOH), cyano (i.e., —CN),dihydrocarbylamido (i.e., —C(═O)—N(Hy)(Hy)), dihydrocarbylamino (i.e.,—N(Hy)(Hy)), di(hydrocarbyl)phosphido, formyl (i.e., —C(═O)H),hydrocarboyl (i.e., —C(═O)—Hy), hydrocarboyloxy (i.e., —O—C(═O)—Hy)hydrocarbylamino (i.e., —NH-Hy), hydrocarbyloxy (i.e., —O-Hy),hydrocarbyloxycarbonyl (i.e., —C(═O)—O-Hy) hydrocarbylsiloxy,hydrocarbylsilylamino, hydrocarbylsulfido (i.e., —S-Hy),hydrocarbylthio, hydrocarbylamido (i.e., —C(═O)—N(H)(Hy)),isothiocyanate, N-heterocycle, perfluorohydrcarbyl, thiocyanate, andhydrocarbyl substituted with one or more groups selected fromalkylamino, amino, aminosulfinyl, aminosulfonyl, azido, dialkylamino,halogen, heteroalkyl, heteroaryl, hydrazinyl, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido;hydrocarbyl is one or more of the following: alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylene, and aryl, where alkyl, alkenyl and alkynyl isoptionally substituted with one or more Hy¹ groups selected fromcycloalkyl, cycloalkylene and aryl, where each Hy¹ group is optionallysubstituted with one or more Hy² groups selected from alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkylene, and aryl; and cycloalkyl,cycloalkylene and aryl is optionally substituted with one or more Hy²groups, provided that when Hy² is selected from alkyl, alkenyl oralkynyl, then Hy² may be substituted with one or more Hy³ groupsselected from cycloalkyl, cycloalkylene and aryl, where each Hy³ groupis optionally substituted with one or more Hy⁴ groups selected fromalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylene, and aryl, and whenHy² is selected from cycloalkyl, cycloalkylene and aryl then Hy² isoptionally substituted with one or more Hy⁴ groups, and further providedthat aryl includes an aryl ring fused to a non-aromatic hydrocarbocyclicring; R¹ at each occurrence is hydrogen; R⁴ is hydrogen; R⁴ is C₁-C₈hydrocarbyl; R² is hydrogen; R² is selected from lower alkyl and lowerhaloalkyl; R² is amino; R² is heterocycle; R² is N-heterocycle; R² ishydrocarbyl; R³ is hydrogen; R³ is selected from phenyl and substitutedphenyl; R³ is phenyl substituted with one or more substituents selectedfrom amino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen,heteroalkyl, heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl,nitro, nitroso, phosphate, phosphinate, phosphonate, phosphonium,phosphorothioate, phosphoryl, sulfamoyl, sulfate, sulfinic acid,sulfonamido, sulfonate, sulfonic acid, sulfonyl, sulfoxido, thiol,thioureido, and ureido; R³ is phenyl substituted with one or moresubstituents selected from hydroxyl, lower alkoxy, and lower alkyl; R³is heteroalkyl; R³ is selected from amino, hydrocarbylamino anddihydrocarbylamino; R³ is hydrocarbylamino where hydrocarbyl is aralkyl;R³ is hydrocarbylamino where hydrocarbyl is alkyl; R³ is amino; R³ ishydrocarbyl. Optionally, one or more of the following compounds areexcluded from the scope of compound useful in the pharmaceuticalcompositions of the present invention: 1H-pyrazole-3,5-diamine,4-(2-benzothiazolyl); 1H-pyrazole-3,5-diamine,4-(2-benzothiazolyl)-N-3-(4-methylphenyl); 1H-pyrazole-3,5-diamine,4-(2-benzothiazolyl)-N-3-phenyl; and 3H-pyrazol-3-one,4-(2-benzothiazolyl)-1,2-dihydro-5-(4-nitrophenyl).

The present invention also provides compounds that may be used in themethods disclosed herein. In one aspect, the present invention providesa compound of formula (1)

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ and R² at each occurrence is independently selected fromamino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R³ isheterocycle; and R⁴ is selected from hydrogen, heteroalkyl, heteroaryl,and hydrocarbyl. In various optional embodiments of this aspect of thepresent invention, one or more of the following criteria may be used todescribe the compounds, where any two or more of the criteria may becombined in describing a group of compounds, however if two criteria areinconsistent then those inconsistent criteria may only be combined inthe alternative: R¹ is hydrogen at each of 4 occurrences; R¹ is hydrogenat 3 out of 4 occurrences; R¹ is hydrogen at 2 out of 4 occurrences; R²is amino; R² is heteroalkyl; R² is heteroaryl; R² is hydrocarbyl; R² ishydrogen; R² is hydroxyl; R³ is N-heterocycle, i.e., the heterocyclicring at R³ contains nitrogen as a ring atom, and this nitrogen atom isdirectly bonded to the pyrazole ring; R³ contains 1-3 heteroatomsselected from O, S and N, and 1-5 carbon atoms; R⁴ is hydrogen; R⁴ ishydrocarbyl; R⁴ is alkyl; R⁴ is C₁-C₈ alkyl.

In another aspect, the present invention provides a compound of formula(1):

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ each occurrence is independently selected from amino,aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R² isamino; R³ is selected from hydrocarbyl, —O-hydrocarbyl and—S-hydrocarbyl; and R⁴ is selected from hydrogen, heteroalkyl,heteroaryl, and hydrocarbyl. In various optional embodiments of thisaspect of the present invention, one or more of the following criteriamay be used to describe the compounds, where any two or more of thecriteria may be combined in describing a group of compounds, however iftwo criteria are inconsistent then those inconsistent criteria may onlybe combined in the alternative: R¹ is hydrogen at each of 4 occurrences;R¹ is hydrogen at 3 out of 4 occurrences; R¹ is hydrogen at 2 out of 4occurrences; R³ is hydrocarbyl; R³ is —O-hydrocarbyl; R³ is—S-hydrocarbyl; the hydrocarbyl portion of R³ is selected from one ormore of the following, where any two or more of the following radicalsmay be combined in order to form a group from which the hydrocarbylportion of R³ is selected: alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkylene, and aryl, where alkyl, alkenyl and alkynyl is optionallysubstituted with one or more Hy¹ groups selected from cycloalkyl,cycloalkylene and aryl, where each Hy¹ group is optionally substitutedwith one or more Hy² groups selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylene, and aryl; and cycloalkyl, cycloalkylene andaryl is optionally substituted with one or more Hy² groups, providedthat when Hy² is selected from alkyl, alkenyl or alkynyl, then Hy² maybe substituted with one or more Hy³ groups selected from cycloalkyl,cycloalkylene and aryl, where each Hy³ group is optionally substitutedwith one or more Hy⁴ groups selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylene, and aryl, and when Hy² is selected fromcycloalkyl, cycloalkylene and aryl then Hy² is optionally substitutedwith one or more Hy⁴ groups, and further provided that aryl includes anaryl ring fused to a non-aromatic hydrocarbocyclic ring; R⁴ is hydrogen;R⁴ is heteroalkyl; R⁴ is heteraryl; R⁴ is hydrocarbyl; R⁴ is alkyl; R⁴is C₁-C₈ alkyl. Optionally, in this aspect of the invention, thecompounds of the invention exclude 1H-pyrazol-5-amine,4-(2-benzothiazolyl)-1,3-diphenyl.

In another aspect, the present invention provides compounds of formula(1):

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ and R² at each occurrence is independently selected fromamino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R³ ishydrogen; and R⁴ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl. In various optional embodiments of this aspect of thepresent invention, one or more of the following criteria may be used todescribe the compounds, where any two or more of the criteria may becombined in describing a group of compounds, however if two criteria areinconsistent then those inconsistent criteria may only be combined inthe alternative: R¹ is hydrogen at each of 4 occurrences; R¹ is hydrogenat 3 out of 4 occurrences; R¹ is hydrogen at 2 out of 4 occurrences; R²is amino; R² is heteroalkyl; R² is phenyl; R² is substituted phenyl; R²is phenyl substituted with one or more substituents selected from anytwo or more of the following: amino, aminosulfinyl, aminosulfonyl, aryl,azido, halogen, heteroalkyl, heteroaryl, hydrazinyl, hydrocarbyl,hydrogen, hydroxyl, nitro, nitroso, phosphate, phosphinate, phosphonate,phosphonium, phosphorothioate, phosphoryl, sulfamoyl, sulfate, sulfinicacid, sulfonamido, sulfonate, sulfonic acid, sulfonyl, sulfoxido, thiol,thioureido, and ureido; R² is heteroaryl; R² is hydrocarbyl; R² ishydrogen; R² is hydroxyl; R⁴ is hydrogen; R⁴ is hydrocarbyl; R⁴ isalkyl; R⁴ is C₁-C₈ alkyl. Optionally, one or more of the followingcompounds are excluded from the scope of compounds within this aspect ofthe present invention: 3H-pyrazol-3-one,4-(2-benzothiazolyl)-2,4-dihydro; 1,3-benzenediol,4-[4-(2-benzothiazolyl)-1H-pyrazol-3-yl]; 1,3-benzenediol,4-[4-(2-benzothiazolyl)-1H-pyrazol-3-yl]-6-ethyl-1-methanesulfonate;benzothiazole, 2-(1H-pyrazol-4-yl); phenol,2-[4-(2-benzothiazolyl)-1H-pyrazol-3-yl]-5-methoxy-4-propyl; phenol,2-[4-(2-benzothiazolyl)-1H-pyrazol-3-yl]5-[(4-nitrophenyl)methoxy];1H-pyrazol-3-amine, 4-(2-benzothiazolyl); 1,3-benzenediol,4-[4-(2-benzothiazolyl)-1H-pyrazol-3-yl]-6-ethyl; 1H-pyrazol-5-amine,4-(2-benzothiazolyl)-1-phenyl; and benzothiazole,2-(1-phenyl-1H-pyrazol-4-yl).

In another aspect, the present invention provides a compound of formula(1):

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ and R² at each occurrence is independently selected fromamino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R³ ishydrocarbyl; and R⁴ is selected from hydrogen, heteroalkyl, heteroaryl,and hydrocarbyl. In various optional embodiments of this aspect of thepresent invention, one or more of the following criteria may be used todescribe the compounds, where any two or more of the criteria may becombined in describing a group of compounds, however if two criteria areinconsistent then those inconsistent criteria may only be combined inthe alternative: R¹ is hydrogen at each of 4 occurrences; R¹ is hydrogenat 3 out of 4 occurrences; R¹ is hydrogen at 2 out of 4 occurrences; R²is hydrogen; R² is hydrocarbyl; R² is heteroalkyl; R² is hydroxyl; R² isheteroaryl; R² is amino; R² is phenyl; R² is substituted phenyl, wherethe substituents is selected from a group that consists of any two ormore of the following: amino, aminosulfinyl, aminosulfonyl, aryl, azido,halogen, heteroalkyl, heteroaryl, hydrazinyl, hydrocarbyl, hydrogen,hydroxyl, nitro, nitroso, phosphate, phosphinate, phosphonate,phosphonium, phosphorothioate, phosphoryl, sulfamoyl, sulfate, sulfinicacid, sulfonamido, sulfonate, sulfonic acid, sulfonyl, sulfoxido, thiol,thioureido, and ureido; R³ (and, independently, R² when R² ishydrocarbyl) is selected from one or more of the following, where anytwo or more of the following radicals may be combined in order to form agroup from which the hydrocarbyl is selected: alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylene, and aryl, where alkyl, alkenyl and alkynyl isoptionally substituted with one or more Hy¹ groups selected fromcycloalkyl, cycloalkylene and aryl, where each Hy¹ group is optionallysubstituted with one or more Hy² groups selected from alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkylene, and aryl; and cycloalkyl,cycloalkylene and aryl is optionally substituted with one or more Hy²groups, provided that when Hy² is selected from alkyl, alkenyl oralkynyl, then Hy² may be substituted with one or more Hy³ groupsselected from cycloalkyl, cycloalkylene and aryl, where each Hy³ groupis optionally substituted with one or more Hy⁴ groups selected fromalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylene, and aryl, and whenHy² is selected from cycloalkyl, cycloalkylene and aryl then Hy² isoptionally substituted with one or more Hy⁴ groups, and further providedthat aryl includes an aryl ring fused to a non-aromatic hydrocarbocyclicring; R⁴ is hydrogen; R⁴ is heteroalkyl; R⁴ is heteraryl; R⁴ ishydrocarbyl; R⁴ is alkyl; R⁴ is C₁-C₈ alkyl. Optionally, one or more ofthe following compounds may be excluded from the scope of compoundswithin this aspect of the present invention: 1H-pyrazol-5-amine,4-(2-benzothiazolyl)-1,3-diphenyl; 1,3-benzenediol,4-[4-(2-benzothiazolyl)-5-methyl-1H-pyrazol-3-yl]-6-propyl; phenol,2-[4-(2-benzothiazolyl)-5-methyl-1H-pyrazol-3-yl]-4-ethyl-5-methoxy;1,3-benzenediol,4-[4-(2-benzothiazolyl)-5-methyl-1H-pyrazol-3-yl]-2-methyl;benzothiazole, 2-(1,3-dimethyl-5-phenyl-1H-pyrazol-4-yl);1,3-benzenediol,4-[4-(2-benzothiazolyl)-5-methyl-1H-pyrazol-3-yl]-6-ethyl;1,3-benzenediol, 4-[4-(2-benzothiazolyl)-5-methyl-1H-pyrazol-3-yl;1,3-benzenediol, and4-(4-(2-benzothiazolyl)-5-methyl-1H-pyrazol-3-yl]-6-ethyl-2[(1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-yl)methyl.

In another aspect, the present invention provides compounds of formula(1)

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹, R² and R³ at each occurrence is independently selectedfrom amino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen,heteroalkyl, heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl,nitro, nitroso, phosphate, phosphinate, phosphonate, phosphonium,phosphorothioate, phosphoryl, sulfamoyl, sulfate, sulfinic acid,sulfonamido, sulfonate, sulfonic acid, sulfonyl, sulfoxido, thiol,thioureido, and ureido, with the proviso that R¹ is not hydrogen in atleast one occurrence of R¹; and R⁴ is selected from hydrogen,heteroalkyl, heteroaryl, and hydrocarbyl. In various optionalembodiments of this aspect of the present invention, the compounds offormula (1) may be described as meeting one or more of the followingcriteria, where any two or more of these criteria may be combined indescribing a group of compounds of formula (1): heteroalkyl is one ormore of the following: aminohydrocarboyl (i.e., —NH—C(═O)—Hy), amido(i.e., —C(═O)—NH₂), carboxylic acid (i.e., —COOH), cyano (i.e., —CN),dihydrocarbylamido (i.e., —C(═O)—N(Hy)(Hy)), dihydrocarbylamino (i.e.,—N(Hy)(Hy)), di(hydrocarbyl)phosphido, formyl (i.e., —C(═O)H),hydrocarboyl (i.e., —C(═O)—Hy), hydrocarboyloxy (i.e., —O—C(═O)—Hy)hydrocarbylamino (i.e., —NH-Hy), hydrocarbyloxy (i.e., —O-Hy),hydrocarbyloxycarbonyl (i.e., —C(═O)—O-Hy) hydrocarbylsiloxy,hydrocarbylsilylamino, hydrocarbylsulfido (i.e., —S-Hy),hydrocarbylthio, hydrocarbylamido (i.e., —C(═O)—N(H)(Hy)),isothiocyanate, N-heterocycle, perfluorohydrcarbyl, thiocyanate, andhydrocarbyl substituted with one or more groups selected fromalkylamino, amino, aminosulfinyl, aminosulfonyl, azido, dialkylamino,halogen, heteroalkyl, heteroaryl, hydrazinyl, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido;hydrocarbyl is one or more of the following: R¹ is selected from amino,aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitrosoand thiol; in at least one occurrence, R¹ is amino, or R¹ isaminosulfinyl, or R¹ is aminosulfonyl, or R¹ is aryl, or R¹ is azido, orR¹ is halogen, or R¹ is heteroalkyl, or R¹ is heteroaryl, or R¹ ishydrazinyl, or R¹ is hydrocarbyl, or R¹ is hydrogen, or R¹ is hydroxyl,or R¹ is nitro, or R¹ is nitroso, or R¹ is thiol; R² is hydrogen; R² isselected from lower alkyl and lower haloalkyl; R² is amino; R² isheterocycle; R² is N-heterocycle; R² is hydrocarbyl; R³ is hydrogen; R³is selected from phenyl and substituted phenyl; R³ is phenyl substitutedwith one or more substituents selected from amino, aminosulfinyl,aminosulfonyl, aryl, azido, halogen, heteroalkyl, heteroaryl,hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso, phosphate,phosphinate, phosphonate, phosphonium, phosphorothioate, phosphoryl,sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate, sulfonicacid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R³ is phenylsubstituted with one or more substituents selected from hydroxyl, loweralkoxy, and lower alkyl; R³ is heteroalkyl; R³ is selected from amino,hydrocarbylamino and dihydrocarbylamino; R³ is hydrocarbylamino wherehydrocarbyl is aralkyl; R³ is hydrocarbylamino where hydrocarbyl isalkyl; R³ is amino; R³ is hydrocarbyl; R⁴ is hydrogen; and R⁴ is C₁-C₈hydrocarbyl.

In another aspect, the present invention provides compounds of formula(1)

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ and R² at each occurrence is independently selected fromamino, aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R³ ishalogen-substituted hydrocarbyl; and R⁴ is selected from hydrogen,heteroalkyl, heteroaryl, and hydrocarbyl. In optional embodiments ofthis aspect of the present invention, one or more of the followingcriteria may be used to describe the compounds, where any two or more ofthe following criteria may be combined: R¹ is hydrogen at each of 4occurrences; R¹ is hydrogen at 3 out of 4 occurrences; R¹ is hydrogen at2 out of 4 occurrences; R² is amino; R² is heteroalkyl; R² is phenyl; R²is substituted phenyl; R² is phenyl substituted with one or moresubstituents selected from any two or more of the following: amino,aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R² isheteroaryl; R² is hydrocarbyl; R² is hydrogen; R² is hydroxyl; R³ istrifluoromethyl; R³ comprises 1 fluorine; R³ comprises 2 fluorines, R³comprises 3 fluorines; R³ is perfluorinated; R⁴ is hydrogen; R⁴ ishydrocarbyl; R⁴ is alkyl; R⁴ is C₁-C₈ alkyl.

In another aspect, the present invention provides compounds of formula(2)

as a single tautomer, a mixture of tautomers, a single stereoisomer, amixture of stereoisomers, or a racemic mixture; or a pharmaceuticallyacceptable salt or solvate thereof; wherein: X is selected from S, O andNR⁹; R⁹ is selected from hydrogen, heteroalkyl, heteroaryl, andhydrocarbyl; R¹ at each occurrence is independently selected from amino,aminosulfinyl, aminosulfonyl, aryl, azido, halogen, heteroalkyl,heteroaryl, hydrazinyl, hydrocarbyl, hydrogen, hydroxyl, nitro, nitroso,phosphate, phosphinate, phosphonate, phosphonium, phosphorothioate,phosphoryl, sulfamoyl, sulfate, sulfinic acid, sulfonamido, sulfonate,sulfonic acid, sulfonyl, sulfoxido, thiol, thioureido, and ureido; R⁴ isselected from hydrogen, heteroalkyl, heteroaryl, and hydrocarbyl; andR⁵, R⁶, R⁷ and R⁸ at each occurrence is independently selected fromheteroalkyl, heteroaryl, hydrocarbyl and hydrogen, with the proviso thatR⁷ and R⁸ may join together to form a heterocyclic ring including thenitrogen to which they are both bonded. In optional embodiments of theinvention, one or more of the following criteria may be used to describethe compound in this aspect of the invention, where any two or more ofthese criteria may be combined: R¹ is hydrogen at each occurrence; R¹excludes hydrogen at one occurrence; R¹ excludes hydrogen at twooccurrences; R⁴ is hydrogen; R⁴ is hydrocarbyl; R⁴ is C₁-C₈ hydrocarbyl;R⁴ is alkyl; R⁴ is C₁-C₈alkyl; R⁵ is hydrogen; R⁶ is hydrogen; R⁷ ishydrogen; R⁸ is hydrocarbyl; R⁸ is heteroalkyl; and R⁸ is heteroaryl.Optionally, one or more of the following compounds is excluded from thescope of this aspect of the present invention: 1H-pyrazole-3,5-diamine,4-(2-benzothiazolyl); 1H-pyrazole-3,5-diamine,4-(2-benzothiazolyl)-N-3-(4-methylphenyl); and 1H-pyrazole-3,-5-diamine,4-(2-benzothiazolyl)-N-3-phenyl.

In each of the compounds and compositions and methods of the presentinvention, in one aspect, X is S (i.e., the compound is a benzothiazolecompound). In each of the compounds, compositions and methods of thepresent invention, in one aspect, X is O (i.e., the compound is abenzooxozole compound). In each of the compounds, compositions andmethods of the present invention, in one aspect X is NR⁹ (i.e., thecompound is a benzoimidazole compound) where R⁹ is selected fromhydrogen, heteroalkyl, heteroaryl, and hydrocarbyl, and in variousoptional embodiments of this aspect of the invention, R⁹ is hydrogen, orR⁹ is hydrocarbyl, or R⁹ is heteroalkyl, or R⁹ is heteroaryl, e.g., Xmay be —N(H)—.

Preparation of the Compounds of the Invention

It is understood that in the following description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described below the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl),tetrahydropyranyl, benzyl, and the like. Suitable protecting groups foramino, amidino and guanidino include t-butoxycarbonyl,benzyloxycarbonyl, and the like. Suitable protecting groups for mercaptoinclude —C(O)—R (where R is alkyl, aryl or aralkyl), p-methoxybenzyl,trityl and the like. Suitable protecting groups for carboxylic acidinclude alkyl, aryl or aralkyl esters.

Protecting groups may be added or removed in accordance with standardtechniques, which are well-known to those skilled in the art and asdescribed herein.

The use of protecting groups is described in detail in Green, T. W. andP. G. M. Wutz, Protective Groups in Organic Synthesis (1991), 2nd Ed.,Wiley-Interscience. The protecting group may also be a polymer resinsuch as a Wang resin or a 2-chlorotrityl chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of formula (1), as described above inthe Summary of the Invention, may not possess pharmacological activityas such, they may be administered to a mammal with cancer orinflammation and thereafter metabolized in the body to form compounds ofthe invention which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds offormula (1) are included within the scope of the invention.

The following Reaction Schemes illustrate methods to make compounds offormula (1). It is understood that one of ordinary skill in the artwould be able to make the compounds of formula (1) by similar methods orby methods known to one skilled in the art. In general, startingcomponents may be obtained from sources such as Aldrich, or synthesizedaccording to sources known to those of ordinary skill in the art (see,e.g., Smith and March, March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure, 5th edition (Wiley Interscience, New York)).Moreover, groups R¹ through R⁵ are selected from components as indicatedin the specification heretofore, and may be attached to startingcomponents, intermediate components, and/or final products according toschemes known to those of ordinary skill in the art. In the followingReaction Schemes, R¹, R², R³, R⁴ and R⁵ are as defined above in theSummary of the Invention and R represents either hydrogen or a loweralkyl group.

Compounds as set forth in compositions and methods of the presentinvention may be prepared by methods disclosed in the literature, and/oras summarized in the following schemes:

In general, compounds of formula (c) (2-(1H-pyrazol-4-yl)benzothiazoles(X═S), benzoxazoles (X═O), benzimidazoles (X═NH)) can be prepared viathe reaction of a substituted 2-aminobenzenethiol, or a 2-aminophenol,or a 1,2-phenyldiamine with a substituted 1H-pyrazole-4-carboxylic acidor a carboxylic acid derivative in an acid such as acetic acid orpolyphosphoric acid at elevated temperature similar to the proceduredescribed in the literature (Shi, D.-F.; Bradshaw, T. D.; Wrigley, S. etal. J. Med. Chem. (1996), 39, 3375). The solution is diluted with waterand neutralized with an ammonia solution. The product is isolated byfiltration or by extraction and, if necessary, is purified by flashchromatography or preparative TLC.

Alternatively, the compounds of this invention can be prepared asdescribed in Reaction Scheme 2.

In general, compounds of formula (c) ((2-(1H-pyrazol-4-yl)benzothiazoles(X═S), (2-(1H-pyrazol-4-yl)benzoxazoles (X═O), and(2-(1H-pyrazol-4-yl)benzimidazoles (X═NH)) can be prepared by couplingof a compound of formula (a) with a substituted 4-iodopyrazole offormula (b) in a solvent such as DMF and in the presence of a transitionmetal catalyst, such as palladium acetate and copper iodide, andtriphenylphosphine and a base such as Cs₂CO₃ as described in theliterature (Pivsa-Art, S.; Satoh, T.; Kawamura, Y. Bull. Chem. Soc.Jpn., (1998) 71, 467).

Alternatively, the compounds of this invention can be prepared asdescribed in Reaction Scheme 3.

In general, compounds of formula (c) ((2-(1H-pyrazol-4-yl)benzothiazoles(X═S), (2-(1H-pyrazol-4-yl)benzoxazoles (X═O), and(2-(1H-pyrazol-4-yl)benzimidazoles (X═NH)) can be prepared through thecoupling of a metallated compound of formula (a) with a substituted4-halopyrazole of formula (b) in the presence of a transition metalcatalyst such as palladium, nickel, or others. The metallated compoundof formula (a) may be prepared by the usual routes known to thoseskilled in the art, such as by metallation using an organometallicreagent, or by metal halogen exchange, or by transmetallation. The metalelement can be boron, zinc, tin, magnesium, lithium or others.

Alternatively, the compounds of this invention can be prepared asdescribed in Reaction Scheme 4.

In general, compounds of formula (c) ((2-(1H-pyrazol-4-yl)benzothiazoles(X═S), (2-(1H-pyrazol-4-yl)benzoxazoles (X═O), and(2-(1H-pyrazol-4-yl)benzimidazoles (X═NH)) can be prepared through thecoupling of a metallated compound of formula (b) with a substituted2-halobenzothiazole in the presence of a transition metal catalyst suchas palladium, nickel, or others. The metallated compound of formula (a)may be prepared by the usual routes known to those skilled in the art,such as by metallation using an organometallic reagent, or by metalhalogen exchange, or by transmetallation. The metal element can beboron, zinc, tin, magnesium, lithium or others.

Alternatively, the compounds of this invention can be prepared asdescribed in Reaction Scheme 5.

In general, compounds of formula (b) are prepared from substitutedacetonitriles of formula (a) reacting with carbon disulfide in thepresence of a base, such as sodium hydride, and an alkylating agent,such as methyl iodide as described in the literature (Augustin, M.;Doelling, W.; J. Prakt. Chem. (1982) 1, 3). The obtained compounds offormula (b) can then be substituted with a nucleophile of formula (c) asdescribed in the literature (Augustin, M.; Doelling, W. supra;) toafford compounds of formula (d). Compounds of formula (d) can then reactwith hydrazine or a substituted hydrazine in a solvent such as ethanol,THF or dioxane to afford compounds of formula (e) in a similar way asdescribed in literature (Fadda, A. A.; Amer, F. A.; Zaki, M. E. A.;Samir, K. H.; Phosphorus, Sulfur Silicon Relat. Elem. (1999), 155, 59).The compounds can be purified by recrystallization or flashchromatography and can be isolated as free bases or as salts.

Compounds of formula (a) can be prepared according to one of the methodsshown in Reaction Scheme 6.

A 2-nitroaniline of formula (a1) can be diazotized and then be reactedwith potassium thiocyanate and cuprous thiocyanate. The resultingproduct can be reduced in the presence of sodium sulfide to give acompound of formula (a2). The resulting substituted nitrobenzene offormula (a2) can be reduced in the presence of tin (II) chloridedihydrate by refluxing the material in a suitable solvent such asethanol to afford a compound of formula (a3). Alternatively, a compoundof formula (a3) can be prepared by reacting a substituted aniline offormula (a4) with ammonium thiocyanate in acetic acid followed by theaddition of bromine. The resulting product can be hydrolyzed by the useof a suitable source of hydroxide such as a 6M solution of sodiumhydroxide. A compound of formula (a3) can also be produced by thereaction of a substituted 2-chloronitrobenzene of formula (a5) and asolution of sodium disulphide which is generated by dissolving sodiumsulfide nonahydrate in hot ethanol followed by the addition of sulfur.This reaction generates a disulfide which can be reduced by refluxingthe intermediate in the presence of tin (II) chloride dihydrate and 2Nhydrochloric acid to produce a compound of formula (a3).

The compound of formula (a) can then be prepared by treating a compoundof formula (a3) with malononitrile in refluxing ethanol. The product canbe isolated by filtration and purified by recrystallization or flashchromatography.

Alternatively, the compounds of this invention can be prepared asdescribed in Reaction Scheme 7.

In general, compounds of formula (b) are prepared from substitutedacetonitiles of formula (a) reacting with carbon disulfide in thepresence of a base, such as sodium hydride, and an alkylating agent,such as methyl iodide as described in the literature (Augustin, M.;Doelling, W.; supra). The obtained compounds of formula (b) can then besubstituted with a Gringard reagent of formula (c) or with anorganolithium compound to afford compounds of formula (d). Compounds offormula (d) can then react with hydrazine or a substituted hydrazine ina solvent such as ethanol, THF or dioxane to afford compounds of formula(e) in a similar way as described in literature (Fadda, A. A.; Amer, F.A.; Zaki, M. E. A.; Samir, K. H.; Phosphorus, Sulfur Silicon Relat.Elem. (1999), 155, 59). The compounds can be purified byrecrystallization or flash chromatography and can be isolated as freebases or as salts.

Alternatively, the compounds of this invention can be prepared asdescribed in Reaction Scheme 8.

In general, compounds of formula (c) can be prepared from substitutedacetonitriles of formula (a) via the reaction with substitutedorthoester of formula (b) in a media such as acetic anhydride asdescribed in the literature (Bontems, R. J.; Anderson, J. D.; Smee, D.F.; Jin, A.; Alaghamandan, H. A. J. Med. Chem. (1990), 8, 2174).Compounds of formula (c) can then be reacted with hydrazine or asubstituted hydrazine in a solvent such as ethanol, THF or dioxane toafford compounds of formula (d). The products can be purified byrecrystallization or flash chromatography and may be isolated as a freebase or as a salt.

Alternatively, the compounds of this invention can be prepared asdescribed in Reaction Scheme 9.

In general, an acetonitrile of formula (a) can be reacted with anactivated carbonyl of formula (b) in the presence of a suitable basesuch as triethylamine to provide a compound of formula (c). Compounds offormula (c) can then be chlorinated to give compounds of formula (d) byreacting the material in neat phosphorous oxychloride at 100° C.Additionally, compounds of formula (d) can be produced by the reactionof compounds of formula (c) with triphenylphosphine, either neat orbound to a resin, and carbon tetrachloride in the presence of a suitablebase such as triethylamine. Compounds of formula (e) can then beprepared from compounds of formula (d) by the reaction with hydrazine ora substituted hydrazine in a solvent such as ethanol, THF or dioxane.

Alternatively, the compounds of this invention can be prepared asdescribed in Reaction Scheme 10.

In general, compounds of formula (c) can be prepared by reacting theappropriate compound of formula (a) with a base such as n-BuLi andtreating the resulting anion with an ester of formula (b) in a suitableaprotic solvent such as THF (Fogagnolo, M.; Giovannini, P. P.; Guerrini,A.; Medici, A.; Pedrini, P.; Colombi, N. Tetrahedron Asymmetry, (1998)9, 2317). The resulting intermediate was condensed with DMF dimethylacetal and hydrazine, as its hydrate or acid salt, or an appropriatelysubstituted hydrazine in a solvent such as ethanol, THF or dioxane toproduce the desired product (as described for the preparation of4-Benzothiazol-2-yl-2H-pyrazol-3-ylamine in Dawood, K. M.; Kandeel, Z.E.; Farag, A. M. J. Chem. Res. Synop. (1998), 4, 208).

Preparation of specific compounds of the invention is described in moredetail below in the Examples

Pharmaceutical Formulations

The compounds of this invention can be incorporated into a variety offormulations for therapeutic administration. More particularly, thecompounds of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate pharmaceutically acceptablecarriers or diluents, and may be formulated into preparations in solid,semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants,gels, microspheres, and aerosols. As such, administration of thecompounds can be achieved in various ways, including oral, buccal,rectal, parenteral, intraperitoneal, intradermal, transdermal,intrathecal, etc., administration. The active agent may be systemicafter administration or may be localized by the use of regionaladministration, intramural administration, or use of an implant thatacts to retain the active dose at the site of implantation.

In pharmaceutical dosage forms, the compounds may be administered in theform of their pharmaceutically acceptable salts. They may also be usedin appropriate association with other pharmaceutically active compounds.The following methods and excipients are merely exemplary and are in noway limiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well tolerated bythe host. The implant containing the inhibitory compounds is placed inproximity to the site of the tumor, so that the local concentration ofactive agent is increased relative to the rest of the body.

The term “unit dosage form”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

The combined use of the provided inhibitory compounds and othercytotoxic agents has the advantages that the required dosages for theindividual drugs is lower, and the effect of the different drugscomplementary. Depending on the patient and condition being treated andon the administration route, the subject inhibitory compounds may beadministered in dosages of 0.1 μg to 10 mg/kg body weight per day. Therange is broad, since in general the efficacy of a therapeutic effectfor different mammals varies widely with doses typically being 20, 30 oreven 40 times smaller (per unit body weight) in man than in the rat.Similarly the mode of administration can have a large effect on dosage.Thus for example oral dosages in the rat may be ten times the injectiondose. Higher doses may be used for localized routes of delivery.

A typical dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

For use in the subject methods, the subject compounds may be formulatedwith other pharmaceutically active agents, particularly otheranti-metastatic, anti-tumor or anti-angiogenic agents. Angiostaticcompounds of interest include angiostatin, endostatin, carboxy terminalpeptides of collagen alpha (XV), etc. Cytotoxic and cytostatic agents ofinterest include adriamycin, alkeran, Ara-C, BICNU, busulfan, CNNU,cisplatinum, cytoxan, daunorubicin, DTIC, 5-FU, hydrea, ifosfamide,methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard,velban, vincristine, vinblastine, VP-16, carboplatinum, fludarabine,gemcitabine, idarubicin, irinotecan, leustatin, navelbine, taxol,taxotere, topotecan, etc.

Methods of Use

The subject compounds are administered to a subject having ahyperproliferative disorders, e.g. to inhibit tumor growth, to inhibitangiogenesis, to decrease inflammation associated with alymphoproliferative disorder, to inhibit graft rejection, orneurological damage due to tissue repair, etc. The present compounds areuseful for prophylactic or therapeutic purposes. As used herein, theterm “treating” is used to refer to both prevention of disease, andtreatment of pre-existing conditions. The prevention of proliferation isaccomplished by administration of the subject compounds prior todevelopment of overt disease, e.g., to prevent the regrowth of tumors,prevent metastatic growth, diminish restenosis associated withcardiovascular surgery, etc. Alternatively the compounds are used totreat ongoing disease, by stabilizing or improving the clinical symptomsof the patient.

The host, or patient, may be from any mammalian species, e.g. primatesp., particularly humans; rodents, including mice, rats and hamsters;rabbits; equines, bovines, canines, felines; etc. Animal models are ofinterest for experimental investigations, providing a model fortreatment of human disease.

The susceptibility of a particular cell to treatment with the subjectcompounds may be determined by in vitro testing. Typically a culture ofthe cell is combined with a subject compound at varying concentrationsfor a period of time sufficient to allow the active agents to inducecell death or inhibit migration, usually between about one h and oneweek. For in vitro testing, cultured cells from a biopsy sample may beused. The viable cells left after treatment are then counted.

The dose will vary depending on the specific compound utilized, specificdisorder, patient status, etc. Typically a therapeutic dose will besufficient to substantially decrease the undesirable cell population inthe targeted tissue, while maintaining patient viability. Treatment willgenerally be continued until there is a substantial reduction, e.g. atleast about 50%, decrease in the cell burden, and may be continued untilthere are essentially none of the undesirable cells detected in thebody.

The compounds also find use in the specific inhibition of signalingpathway mediated by protein kinases. Protein kinases are involved insignaling pathways for such important cellular activities as responsesto extracellular signals and cell cycle checkpoints. Inhibition ofspecific protein kinases provides a means of intervening in thesesignaling pathways, for example to block the effect of an extracellularsignal, to release a cell from cell cycle checkpoint, etc. Defects inthe activity of protein kinases are associated with a variety ofpathological or clinical conditions, where there is a defect insignaling mediated by protein kinases. Such conditions include thoseassociated with defects in cell cycle regulation or in response toextracellular signals, e.g. hyperglycemia and diabetes Type I and TypeII, immunological disorders, e.g. autoimmune and immunodeficiencydiseases; hyperproliferative disorders, which may include psoriasis,arthritis, inflammation, angiogenesis, endometriosis, scarring, cancer,etc.

The compounds of the present invention are active in inhibiting purifiedkinase proteins, i.e. there is a decrease in the phosphorylation of aspecific substrate in the presence of the compound. A protein kinase ofparticular interest in integrin linked kinase (ILK). ILK is a serinethreonine kinase. The DNA and predicted amino acid sequence may beaccessed at Genbank, no. U40282, or as published in Hannigan et al.Nature (1996) 379:91-96. ILK regulates integrin extracellular activity(ECM interactions) from inside the cell via its direct interaction withthe integrin subunit. Interfering with ILK activity allows the specifictargeting of integrin function, while leaving other essential signalingpathways intact. Increased levels of cellular ILK activity shortcircuits the normal requirement for adhesion to extracellular membranein regulating cell growth. Thus, inhibiting ILK activity may inhibitanchorage-independent cell growth.

It is also known that many cell types undergo apoptosis if theappropriate contacts with extracellular matrix proteins are notmaintained (anoikis). The induction of apoptosis by the subjectcompounds in such cells predicts an association with the ILK signalingpathway.

The compounds of the present invention bind to protein kinases at a highaffinity, and find use as affinity reagents for the isolation and/orpurification of such kinases. Affinity chromatography is used as amethod of separating and purifying protein kinases and phosphatasesusing the biochemical affinity of the enzyme for inhibitors that act onit. The compounds are coupled to a matrix or gel. Preferably amicrosphere or matrix is used as the support. Such supports are known inthe art and commercially available. The inhibitor-coupled support isused to separate an enzyme that binds to the inhibitor from a complexmixture, e.g. a cell lysate, that may optionally be partially purified.The sample mixture is contacted with the inhibitor coupled support underconditions that minimize non-specific binding. Methods known in the artinclude columns, gels, capillaries, etc. The unbound compounds arewashed free of the resin, and the bound proteins are then eluted in asuitable buffer.

The compounds of the invention may also be useful as reagents forstudying signal transduction or any of the clinical disorders listedthroughout this application.

Hyper-Proliferative Disorders of Interest

There are many disorders associated with a dysregulation of cellularproliferation. The conditions of interest include, but are not limitedto, the following conditions.

The subject methods are applied to the treatment of a variety ofconditions where there is proliferation and/or migration of smoothmuscle cells, and/or inflammatory cells into the intimal layer of avessel, resulting in restricted blood flow through that vessel, i.e.neointimal occlusive lesions. Occlusive vascular conditions of interestinclude atherosclerosis, graft coronary vascular disease aftertransplantation, vein graft stenosis, peri-anastomatic prosthetic graftstenosis, restenosis after angioplasty or stent placement, and the like.

Diseases where there is hyperproliferation and tissue remodelling orrepair of reproductive tissue, e.g. uterine, testicular and ovariancarcinomas, endometriosis, squamous and glandular epithelial carcinomasof the cervix, etc. are reduced in cell number by administration of thesubject compounds

Tumor cells are characterized by uncontrolled growth, invasion tosurrounding tissues, and metastatic spread to distant sites. Growth andexpansion requires an ability not only to proliferate, but also todown-modulate cell death (apoptosis) and activate angiogenesis toproduce a tumor neovasculature. Angiogenesis may be inhibited byaffecting the cellular ability to interact with the extracellularenvironment and to migrate, which is an integrin-specific function, orby regulating apoptosis of the endothelial cells. Integrins function incell-to-cell and cell-to-extracellular matrix (ECM) adhesiveinteractions and transduce signals from the ECM to the cell interior andvice versa. Since these properties implicate integrin involvement incell migration, invasion, intra- and extra-vasation, and plateletinteraction, a role for integrins in tumor growth and metastasis isobvious.

Tumors of interest for treatment include carcinomas, e.g. colon,duodenal, prostate, breast, melanoma, ductal, hepatic, pancreatic,renal, endometrial, stomach, dysplastic oral mucosa, polyposis, invasiveoral cancer, non-small cell lung carcinoma, transitional and squamouscell urinary carcinoma etc.; neurological malignancies, e.g.neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhoodacute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia,malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-celllymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoidhyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichenplanus, etc.; and the like.

Some cancers of particular interest include breast cancers, which areprimarily adenocarcinoma subtypes. Ductal carcinoma in situ (DCIS) isthe most common type of noninvasive breast cancer. In DCIS, themalignant cells have not metastasized through the walls of the ductsinto the fatty tissue of the breast. Infiltrating (or invasive) ductalcarcinoma (IDC) has metastasized through the wall of the duct andinvaded the fatty tissue of the breast. Infiltrating (or invasive)lobular carcinoma (ILC) is similar to IDC, in that it has the potentialmetastasize elsewhere in the body. About 10% to 15% of invasive breastcancers are invasive lobular carcinomas.

Also of interest is non-small cell lung carcinoma. Non-small cell lungcancer (NSCLC) is made up of three general subtypes of lung cancer.Epidermoid carcinoma (also called squamous cell carcinoma) usuallystarts in one of the larger bronchial tubes and grows relatively slowly.The size of these tumors can range from very small to quite large.Adenocarcinoma starts growing near the outside surface of the lung andmay vary in both size and growth rate. Some slowly growingadenocarcinomas are described as alveolar cell cancer. Large cellcarcinoma starts near the surface of the lung, grows rapidly, and thegrowth is usually fairly large when diagnosed. Other less common formsof lung cancer are carcinoid, cylindroma, mucoepidermoid, and malignantmesothelioma.

Melanoma is a malignant tumor of melanocytes. Although most melanomasarise in the skin, they also may arise from mucosal surfaces or at othersites to which neural crest cells migrate. Melanoma occurs predominantlyin adults, and more than half of the cases arise in apparently normalareas of the skin. Prognosis is affected by clinical and histologicalfactors and by anatomic location of the lesion. Thickness and/or levelof invasion of the melanoma, mitotic index, tumor infiltratinglymphocytes, and ulceration or bleeding at the primary site affect theprognosis. Clinical staging is based on whether the tumor has spread toregional lymph nodes or distant sites. For disease clinically confinedto the primary site, the greater the thickness and depth of localinvasion of the melanoma, the higher the chance of lymph node metastasesand the worse the prognosis. Melanoma can spread by local extension(through lymphatics) and/or by hematogenous routes to distant sites. Anyorgan may be involved by metastases, but lungs and liver are commonsites.

Other hyperproliferative diseases of interest relate to epidermalhyperproliferation, tissue remodelling and repair. For example, thechronic skin inflammation of psoriasis is associated with hyperplasticepidermal keratinocytes as well as infiltrating mononuclear cells,including CD4+ memory T cells, neutrophils and macrophages.

The proliferation of immune cells is associated with a number ofautoimmune and lymphoproliferative disorders. Diseases of interestinclude multiple sclerosis, rheumatoid arthritis and insulin dependentdiabetes mellitus. Evidence suggests that abnormalities in apoptosisplay a part in the pathogenesis of systemic lupus erythematosus (SLE).Other lymphoproliferative conditions the inherited disorder oflymphocyte apoptosis, which is an autoimmune lymphoproliferativesyndrome, as well as a number of leukemias and lymphomas. Symptoms ofallergies to environmental and food agents, as well as inflammatorybowel disease, may also be alleviated by the compounds of the invention.

In one aspect of the invention, the pyrazolylbenzothiazole compoundsdisclosed herein may be used to inhibit integrin-linked kinase (ILK) forthe treatment of inflammatory diseases and autoimmune conditions such aspsoriasis in which the immune system directly contributes to diseasepathogenesis. Integrin-linked kinase (ILK) is a 59 kDa serine/threoninekinase that associates with the cytoplasmic tail portions of β1 and β3integrins, molecules that mediate adhesion of different cells to othercells or various components of the extracellular matrix. Furthermore,ILK associates with and interacts with a number of intracellularproteins. The enzymatic activity of ILK is modulated by the interactionof ILK-expressing cells with the extracellular matrix (ECM) componentfibronectin, integrin clustering as well as a variety of growth factors.ILK activity is associated with a number of downstream signaling events.Upon adhesion to ECM, integrins and a selective group of cytoskeletaland signaling proteins are recruited to cell matrix contact sites wherethey serve to link the actin cytoskeleton to the ECM. These linksfunction to mediate communication between the intracellular andextracellular compartments.

Thus, in one aspect the present invention relates to therapeuticcompositions and methods for the treatment of inflammatory disordersincluding autoimmune diseases using compounds that inhibit ILK activity.Such disorders and diseases include, but are not limited to, psoriasis,rheumatoid arthritis, multiple sclerosis, scleroderma, systemic lupuserythematosus, Sjögren's syndrome, atopic dermatitis, asthma, andallergy. Target cells susceptible to the treatment include cellsinvolved in instigating autoimmune reactions as well as those sufferingor responding from the effects of autoimmune attack or inflammatoryevents.

As mentioned above, pyrazolylbenzothiazole compounds that function asILK inhibitors may be formulated into a variety of compositions.Suitable excipients for use with ILK inhibitors include water, saline,dextrose, glycerol, Cremaphor™, ethanol and the like. These compositionsmay comprise further components such as conventional delivery agents andexcipients including isotonising agents, pH regulators, solvents,solubilizers, dyes, gelling agents, thickeners, buffers and combinationsthereof. To ameliorate inflammatory/autoimmune diseases such aspsoriasis, inhibitors of ILK are administered by an appropriate meansincluding, but not limited to, oral, intravenous, subcutaneous,intramuscular or topical routes. The local delivery, such as topical, ofan ILK inhibitor provides high concentrations at the treatment sitewhile lowering the likelihood of unwanted non-specific or otherundesirable effects that might be associated with systemic delivery ofsuch compounds. For the local delivery of pyrazolylbenzothiazolecompounds for psoriasis and other cutaneous inflammatory or autoimmuneconditions, the compounds may be administered in excipients containingconcentrations of about 0.01 to about 10 mg/ml directly applied to theskin. If systemic delivery is required, a dose range of 0.1 mg/kg to 100mg/kg body weight, preferably less than 10 mg/kg, is administered. Thepyrazolylbenzothiazole compound may be given up to 3 times daily. Oraldelivery may be given in tablets, capsules, liquid suspensions orsolutions.

Although psoriasis is not life threatening, the social stigma andreduction in quality of life associated with disease are profound issuesfor these patients and their families. Established anti-psoriasistherapies have been grouped into suppressive and remittive types.Suppressive therapies (e.g. cyclosporine, topical calcitriol,methotrexate, retinoids), produce plaque clearance although thesemedications are not associated with a complete normalization of skinpharmacodynamic markers or large reductions in plaque T cell numbers.Phototherapy with ultraviolet (UV) B (280-320 nm) light alone or incombination with coal tar derivatives and photochemotherapy with8-methoxypsoralen combined with UVA (320-400 nm) light (PUVA) areclassified as remittive-type anti-psoriasis therapies. UVB light andPUVA are typically delivered in multiple treatment sessions, oftenseveral times weekly, until plaque clearance is achieved. The presentinvention provides pyrazolylbenzothiazole compounds that may beadministered in combination with established anti-psoriasis therapies.

Renal Disorders

In one aspect of the invention, the pyrazolylbenzothiazole compoundsdisclosed herein may be used to modulate integrin-linked kinase (ILK)for the treatment of renal diseases. Thus, the present inventionprovides therapeutic compositions and methods for treating renaldisease, and specifically provides therapeutic compositions and methodsdirected to modulating, and especially inhibiting, the activity of ILKso as to ameliorate glomerular renal disease states which may result inproteinuria, or states characterized by tubular or tubulo-interstitialdamage. Preferred pyrazolylbenzothiazole compounds may be identified byscreening for biological activity in an ILK-based functional assay, e.g.in vitro or in vivo ILK kinase activity.

According to current therapies, chronic progression of renal disease canbe slowed for 6-12 months using angiotensin-converting enzyme (ACE)inhibitors, but there is no other satisfactory treatment at this timebesides dialysis and ultimately transplantation of the organ. Accordingto the present invention, pyrazolylbenzothiazole compounds may beadministered at an appropriate time, before, concurrent or after, inrelation to a second therapy for treating renal disorder, where thatsecond therapy includes, but is not limited to, administration of an ACEinhibitor, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier to a mammal in need thereof. Aceinhibitors include, but are not limited to, captopril, benazepril,enalapril, fosinopril, lisinopril, quinapril, ramipril, imidapril,perindopril, erbumine, and trandolapril. ACE Receptor Blockers may alsobe used in place of, or as well as, ACE inhibitors, and these includelosartan, irbesartan, candesartan, cilexetil, and valsartan.

Thus, in one aspect, the present invention provides a method fortreating a patient with renal dysfunction comprising administering tothe patient an effective amount of a pyrazolylbenzothiazole compound orcomposition including a pyrazolylbenzothiazole compound as disclosedherein. In various embodiments, the compound is administered orally, orthe compound is administered intravenously, or the compound isadministered intraperitoneally. The compound may be administeredintralumenally in or around the kidney. The patient may also be treatedwith an ACE inhibitor.

In one aspect, the present invention provides a method for lowering theprotein levels in urine, comprising administering to that patient aneffective amount of a pyrazolylbenzothiazole compound, or compositioncontaining an pyrazolylbenzothiazole compound as disclosed herein. Invarious embodiments, the compound is administered orally, orintravenously, or intraperitoneally. The compound may be administeredintralumenally in or around the kidney. The patient may also be treatedwith an ACE inhibitor.

Eye Disorders

In one aspect, the present invention relates to the use ofpyrazolylbenzothiazoles as disclosed herein in the treatment of variouseye diseases with underlining pathology of neovascularization of cornea,iris, retina or choroids. The subject methods are used for prophylacticor therapeutic purposes to treat ocular diseases to prevent, reduce orreverse the loss of visual acuity as well as loss of vision secondary toneovascularization of cornea, iris, retina or choroid. The term“treating” is used to refer to both prevention of disease, and treatmentof pre-existing conditions. While treatment during early stages isdesirable, the adverse symptoms of the disease may be at least partiallyalleviated by treatment during later stages

In one aspect, pyrazolylbenzothiazole compounds that modulate theactivity of integrin linked kinase (ILK) are administered systemicallyor locally to treat ophthalmic diseases with an underlining pathologythat is characteristic of ocular neovascularization. Such a treatment isused alone as single therapy or in combination with a second therapy asan adjunct to prevent, to reduce or to reverse the loss of visual acuityas well as loss of vision secondary to neovascularization of cornea,iris, retina or choroids.

For example, in one aspect the invention is directed to a method toprevent, to reduce or to reverse ocular neovascularization in an eye ofan animal having a neovascular lesion, comprising the steps ofidentifying said lesion in the eye of the animal, administering to theanimal an amount of a pyrazolylbenzothiazole compound as disclosedherein sufficient to allow said compound to localize in said lesion.Methods utilizing local administration that provides for a prolongedlocalized concentration, which may utilize sustained release implants,viscous solutions, or other topical formulation, are of particularinterest. A pyrazolylbenzothiazole compound can be administered alone assingle therapy, or in combination with a second therapy, for example atan appropriate time, before, concurrent or after, in relation to asecond therapy including but not limited to Visudyne™ therapy,photocoagulation or transpupillary thermotherapy as an adjunct treatmentfor ocular neovascularization.

Some examples of ocular disorders that may be treated by variousembodiments of the present invention include, without limitation:retinal diseases (diabetic retinopathy, chronic glaucoma, retinaldetachment, sickle cell retinopathy, age related macular degeneration(AMD) due to subretinal neovascularization); rubeosis iritis;inflammatory diseases; chronic uveitis; neoplasms (retinoblastoma,pseudoglioma); Fuchs' heterochromic iridocyclitis; neovascular glaucoma;corneal neovascularization (inflammatory, transplantation, developmentalhypoplasia of the iris); neovascularization resulting following acombined vitrectomy and lensectomy; vascular diseases (retinal ischemia,choroidal vascular insufficiency, choroidal thrombosis, carotid arteryischemia); neovascularization of the optic nerve; and neovascularizationdue to penetration of the eye or contusive ocular injury.

In practicing the method of treatment or use of a pyrazolylbenzothiazolecompound in an ophthalmic diseases with an underlining pathology that ischaracteristic of ocular neovascularization, a therapeutically effectiveamount of a pyrazolylbenzothiazole compound is administered to a subjectafflicted with a disease or disorder related to neovascularization, orto a tissue that has been neovascularized. The inhibitor may beadministered in accordance with the method of the invention either aloneor in combination with other known therapies for neovascularization.When co-administered with one or more other therapies, thepyrazolylbenzothiazole compound may be administered eithersimultaneously with the other treatment(s), or sequentially. Ifadministered sequentially, the attending physician will decide on theappropriate sequence of administration, which may be before or after asecond therapy.

Secondary therapies of interest include photodynamic therapy, forexample verteporfin (VISUDYNE™) therapy, see, for example Madreperla(2001) Arch Ophthalmol. 119(11):1606-1610; Harding (2001) Eye 15(Pt3):407-12; Sharma (2001) Can Fam Physician 47:955, 963. Photocoagulationor transpupillary thermotherapy, see, e.g., Rogers et al. (2001) CurrOpin Ophthalmol 12(3): 212-5; Ardjomand et al. (2001) Ophthalmologica215(3):241-4; Mainster et al. (2000) Ophthalmic Surg Lasers31(5):359-73. Other therapies include, without limitation, those setforth in U.S. Pat. No. 6,297,228, “Use of angiostatic steroids inphotodynamic therapy”, U.S. Pat. No. 6,271,233 “Method for treatingocular neovascular diseases”; U.S. Pat. No. 6,248,734 “Use ofphotodynamic therapy for prevention of secondary cataracts”; U.S. Pat.No. RE37,180 “Photochemotherapeutical obstruction of newly-formed bloodvessels”; U.S. Pat. No. 6,225,303 “Use of green porphyrins to treatneovasculature in the eye”; U.S. Pat. No. 6,217,895 “Method for treatingand/or preventing retinal diseases with sustained releasecorticosteroids”; U.S. Pat. No. 6,214,819 “Method for treating ocularneovascular diseases”, and the like.

Some eye diseases lend themselves to acute treatment while othersrequire longer term therapy. Proliferative retinopathy can reach athreshold in a matter of days as seen in ROP, some cases of diabeticretinopathy, and neovascular glaucoma. Premature infants are at risk forneovascularization around what would be 35 weeks gestation, a few weeksafter birth, and will remain at risk for a short period of time untilthe retina becomes vascularized. Diabetic retinopathy can be acute butmay also smolder in the proliferative phase for considerably longer.Suitable animal models exist for determination of appropriate dosage,although the efficacy of a therapeutic effect for different mammalsvaries widely, for example doses typically are 20, 30 or even 40 timessmaller (per unit body weight) in man than in the rat. Similarly themode of administration can have a large effect on dosage. A murine modelof oxygen-induced retinal neovascularization has been established whichoccurs in 100% of treated animals and is quantifiable (Smith et al.(1994) Invest. Ophthalmol. Vis. Sci 35:101-111). Bioactivity can bedetermined by methods including the Miles vessel permeability assay(Miles and Miles, J. Physiol. (Lond.) (1952) 118:228), which measuresvessel permeability, and endothelial cell mitogenicity, which measurescell growth.

For local application, a range of about 0.05 to 0.2 or about 0.5 mg/mlof a pyrazolylbenzothiazole compound in an appropriate formulation isadministrated either intra-ocularly (intra-vitreous, subretinal,intra-anterior chamber, intra-scleral), peri-ocularly, or topically ontothe cornea. For systemic application, a range of 0.05 to 100 mg/kg bodyweight, preferably less than about 10 mg/kg is administered to treat eyedisease. For intra- or peri-ocular administration, apyrazolylbenzothiazole compound in an injectable formulation isadministered by either an intra-ocular injection at above-describedconcentrations and at a frequency of once every 2-6 months or by anintra-ocular implantation of a device or a specific formulation of anILK inhibitor allowing sustained release of the ILK inhibitor over aperiod of time. For corneal application, an ILK inhibitor in anappropriate formulation is applied topically onto the cornea at afrequency of once very 4-6 hours. For systemic application, an ILKinhibitor in appropriate formulation is administered orally 1-3 times aday.

Thus, in one aspect, the present invention provides a method fortreating ocular neovascularization, the method comprising administeringa pyrazolylbenzothiazole compound or composition comprising such acompound as described herein to treat ocular neovascularization.Optionally, the treatment reduces or reverses the loss of visual acuitysecondary to neovascularization of cornea, iris, retina or choroid. Themethoer may further comprise administering a second therapy for ocularneovascularization, where a suitable second therapy is selected from thegroup consisting of photodynamic therapy, photocoagulation andtranspupillary thermotherapy. In the present method, the ocularneovascularization may be selected from the group consisting of diabeticretinopathy, chronic glaucoma, retinal detachment, sickle cellretinopathy, age related macular degeneration (AMD) due to subretinalneovascularization; rubeosis iritis; inflammatory diseases; chronicuveitis; neoplasms; Fuchs' heterochromic iridocyclitis; neovascularglaucoma; corneal neovascularization; neovascularization resultingfollowing a combined vitrectomy and lensectomy; retinal ischemia,choroidal vascular insufficiency, choroidal thrombosis, carotid arteryischemia; neovascularization of the optic nerve; and neovascularizationdue to penetration of the eye or contusive ocular injury. In variousembodiments, the pyrazolylbenzothiazole compound is administeredsystemically, or intra-ocularly, or peri-ocularly, or is administeredtopically onto the cornea, or is administered by intra-ocular injection,or is administered by intra-ocular implantation.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric. The starting materials used in the examples can bepurchased from a chemical supplier such as Aldrich and Lancaster or canbe prepared following the preparation procedures described herein:

EXAMPLES Example 1 Preparation 1: Synthesis of2-Amino-4-fluorobenzenethiol

To a solution of 2-chloro-5-fluoronitrobenzene (1.81 g, 10.31 mmol)dissolved in 30 mL of deionized water at room temperature was addedsodium sulfide nonahydrate (9.90 g, 41.24 mmol) in a single portion. Theresulting solution was heated to reflux and stirred under nitrogen for32 hours. The resulting light yellow solution was then cooled to roomtemperature and was washed with 5×50 mL of ethyl acetate. The organicphase was dried over anhydrous magnesium sulfate, filtered, andevaporated to yield a yellow oil. The crude material was purified byflash chromatography eluting with CH₂Cl₂:MeOH=10:1 to yield 0.36 g (25%)of the title compound. MS (m/z, ES+): 144.0 (M+1, 100%); IR (KBr): 3430,3340, 1615, 1573, 1482, 1281, 1248, 1172, 1124, 1044, 975, 840, 792cm⁻¹; ¹H NMR (300 MHz, ppm, DMSO-d₆) δ: 6.89 (dt, 1H), 6.50 (dd, 1H),6.21 (dt, 1H), 5.80 (br s, 2H).

Preparation 2: Synthesis of Thiazolo[5,4-b]pyridin-2-ylamine

A suspension of 2-chloro-3-aminopyridine (3 g, 23 mmole), and ammoniumthiocyanate (3.5 g, 46.5 mmole) in 23 mL of ethanol was acidified withconc. HCl to pH ˜1 (˜1.8 mL). The reaction mixture was heated to 85° C.for 3 days. At this point, the solvent was evaporated and residual waterwas removed aziotropically by the distillation of 2-propanol. The yellowresidue was mixed with 12 mL of 7 M ammonium hydroxide and 7 mL ofchloroform. The solid was isolated by filtration to yield 1.50 g (43%)of the product as a white powder. MS (m/z, ES+): 152 (M+1, 100%).

Preparation 3: Synthesis of 4-Fluorobenzothiazol-2-ylamine

To a suspension of (2-fluorophenyl)thiourea (1.7 g, 10.0 mmol) inchloroform (25 ml) was added a solution of bromine (0.51 mL; 10.0 mmol)dropwise at room temperature. The resulting mixture was heated underreflux for 3 hrs. The solvent was evaporated, water was added and themixture was neutralized with ammonium hydroxide. The resulting whiteprecipitation was collected by filtration and dried to yield 1.2 g (72%)of the title compound which was used in the subsequent step withoutfurther purification.

Preparation 4: Synthesis of 7-Chloro-4-methoxybenzothiazol-2-ylamine

The title compound was prepared from (5-chloro-2-methoxyphenyl)thiourea(2.17 g, 10.0 mmol) using a procedure analogous to that described inPreparation 3. The title compound was isolated in a yield of 2.1 g(98%).

Preparation 5: Synthesis of 4-Amino-2-fluorobenzoic acid

To a solution of 2-fluoro-4-nitrobenzoic acid (1.0 g, 5.4 mmol) in 20 mLof a mixture of acetic acid and methanol (1:1), was added a catalyticamount of palladium on charcoal (25 mg). The reaction was stirred underan atmosphere of hydrogen gas at room temperature overnight. The mixturewas then filtered through celite and the solvent was removed byevaporation to yield 0.86 g (100%) of the title compound as a creamcoloured solid.

Preparation 6: Synthesis of 4-Amino-2-fluoro-N-methylbenzenesulfonamide

A solution of N-(3-fluorophenyl)acetamide (20.0, 0.13 mol) inchlorosulfonic acid (150 mL) was heated to 75° C. for 1 hr. The solutionwas allowed to cool to room temperature and was poured over ice. Theresulting slurry was extracted with dichloromethane. The combinedorganic extracts were dried over magnesium sulfate, filtered andevaporated to yield 15.5 g (47%) of4-acetylamino-2-fluorobenzenesulfonyl chloride as a paste. The crudematerial was used in the subsequent step without further purification.

The above prepared 4-acetylamino-2-fluorobenzenesulfonyl chloride (2 g,7.9 mmol) was dissolved in dichloromethane and methylamine (9.9 mL of a2M solution in THF, 19.9 mmol) was added. The reaction was stirred atroom temperature for 2 hrs. The solvent was then evaporated underreduced pressure and the resulting solid was suspended in 50 mL ofwater. The solid was isolated by filtration and dried to yield 1.55 g(79%) of N-(3-fluoro-4-methylsulfamoylphenyl)acetamide which was used inthe subsequent step without further purification.

The above prepared N-(3-fluoro-4-methylsulfamoylphenyl)acetamide (1.55g, 6.3 mmol) was suspended in 40 mL of 6M HCl and heated to reflux for 1hr. The reaction was cooled in an ice bath and an NaOH solution wasadded to adjust the mixture to pH 5. The resulting white precipitate wasisolated by filtration and was washed with water and dried to yield 1.07g (83%) of the title compound. MS (m/z, ES+): 205.0 (M+1, 100%); ¹H NMR(500 MHz, ppm, DMSO-d₆): 7.35 (m, 1H), 7.12 (m, 1H), 6.40 (m, 2H), 6.23(s, 2H), 2.40 (s, 3H).

Preparation 7: Synthesis of 4-Amino-N-methylbenzenesulfonamide

To an ice cold solution of 4-nitrobenzenesulfonyl chloride (2.7 g, 12mmol) and triethylamine (27 mmol) in 60 mL of dry THF was addedmethylamine (8 mL of a 2 M solution in THF, 16 mmol). The mixture wasstirred at room temperature overnight. Brine was added and the reactionsolution was extracted with ethyl acetate. The combined organic extractswere dried over sodium sulfate, filtered, and evaporated to yield4-nitrobenzenesulfonic acid methylamide as an oil. The crude materialwas used in the subsequent reaction without further purification.

To a solution of the crude 4-nitrobenzenesulfonyl methylamide preparedabove in 45 mL of ethanol, was added a 1 mL slurry of Raney Nickel.Hydrazine monohydrate (18 mmol) was added slowly in several portions. Achange in the solution colour from yellow to colourless indicated thereaction was complete. The mixture was stirred for an additional 1 hr.The solids were removed by filtration and the solvent was evaporated.The resulting crude material was purified by flash column chromatographyeluting with CH₂Cl₂:MeOH=10:1 to yield 2.09 g (90% for two steps) of thetitle compound as a pale orange powder.

Preparation 8: Synthesis of 2-Aminobenzothiazole-7-carboxylic acid

3-Isothiocyanatobenzoic acid (1.0 g, 5.6 mmol) in 40 mL of ethanol wasstirred at room temperature for 1 hr while ammonia gas was bubbledthrough the solution. The resulting white precipitate was isolated byfiltration. The volume of the filtrate was reduced to induce theprecipitation of additional white solid that was also isolated byfiltration. The total yield of 3-thioureidobenzoic acid was 1.06 g(97%).

To a suspension of 3-thioureidobenzoic acid (1.06 g, 5.41 mmol) inchloroform (25 mL) was added a solution of bromine (0.4 mL, 8.0 mmol)dropwise at room temperature. The resulting mixture was heated underreflux for 3 hrs and allowed to stir at room temperature for 60 hrs. Thesolvent was evaporated, water was added and the resulting solids wereisolated by filtration. The crude product was washed with water anddried to yield 1.03 g (98%) of the title compound as a gray solid.

Preparation 9: Synthesis of 2-Cyanomethylbenzothiazole-6-sulfonic acidamide

1. To a suspension of 4-amino-N-methylbenzenesulfonamide (43 g, 250mmol) in 400 mL acetic acid was added ammonium thiocyanate (49 g, 650mmol) in several portions. After the mixture had been stirred at roomtemperature for 30 min, a solution of bromine (13 mL, 250 mmol) in 160mL of acetic acid was added dropwise. The reaction was then stirred atroom temperature for 64 hrs. The resulting solids were isolated byfiltration, washed with saturated NaHCO₃ solution and water, and driedto yield 60 g of material. The filtrate was evaporated to dryness, theresidue was suspended in a saturated NaHCO₃ solution and additionalproduct was extracted into ethyl acetate. The organic extracts weredried over sodium sulfate, filtered, and evaporated to yield anadditional 6.2 g of product to give a total of 66 g of2-aminobenzothiazole-6-sulfonic acid amide.

2. A solution of 2-aminobenzothiazole-6-sulfonic acid amide (66 g, 287mmol) and NaOH (90 g, 2.3 mol) in 400 mL of water was refluxed underargon overnight. The reaction solution was cooled in ice bath and wasacidified to approximately pH 3 by the addition of concentrated HCl(˜180 mL). The resulting precipitate was isolated by filtration anddried to yield 74 g of 4-amino-3-mercaptobenzenesulfonamide as a whitepowder. MS (m/z, ES+): 205 (M+1, 100%).

3. A suspension of 4-amino-3-mercaptobenzenesulfonamide (74 g, 208 mmol)and malononitrile (25 g, 375 mmol) in 650 mL of ethanol with 1.3 mL ofconc. HCl was refluxed under argon overnight. The dark brown solids wereisolated by filtration and were washed with ethanol and ether to yieldapproximately 30 g of the crude product. The volume of the filtrate wasreduced by evaporation and the solids that precipitated were isolated byfiltration to yield an additional 12 g of product. The crude materialwas purified by flash column chromatography eluting withCH₂Cl₂:MeOH:ammonia (60:20:1 mL) to yield a total of 22.2 g (35% forthree steps) of the title compound as an orange powder.

Preparation 10: Synthesis of(5-Fluoro-6-methoxybenzothiazol-2-yl)acetonitrile

The title compound was prepared two steps starting from6-fluoro-5-methoxyaniline (1.41 g, 10 mmol) and ammonium thiocyanate(1.83 g, 24 mmol) using a procedure analogous to that described inPreparation 9. The title compound was isolated in a yield of 240 mg (10%for 3 steps).

Preparation 11: Synthesis of (6-Methoxybenzothiazol-2-yl)acetonitrile

The title compound was prepared two steps starting from6-methoxybenzothiazol-2-ylamine (10 g, 55 mmol) using a procedureanalogous to that described in Preparation 9. The title compound wasisolated in a yield of 8.5 g (75% for 2 steps).

Preparation 12: Synthesis of2-(5-Fluoro-6-methoxybenzolthiazole)-3,3-bis-methylsulfanylacrylonitrile

To a stirred cool solution of2-(5-fluoro-6-methoxybenzolthiazole)acetonitrile, (1.48 g, 6.65 mmol),carbon disulfide (0.90 mL, 15 mmol) and iodomethane (1.7 mL, 27 mmol) inanhydrous dimethylsulfoxide (40 mL) was added sodium hydride (540 mg ofa 60% slurry in mineral oil, 13.5 mmol) in portions under an inertatmosphere. The dark red reaction mixture was stirred at 5° C. for 30minutes and then at room temperature for 2 hrs before it was quenchedwith saturated aqueous ammonium chloride (20 mL) and diluted withdistilled water. The resulting orange precipitate was isolated byfiltration, washed with water, suspended in isopropanol and re-filtered.The precipitate was then dried under vacuum to yield 1.61 g (74%) of thetitle compound as a yellow solid. MS (m/z, ES+): 328 (M+1, 100%).

Preparation 13: Synthesis of2-(6-Methoxybenzolthiazole)-3,3-bis-methylsulfanylacrylonitrile

The title compound was prepared from2-(6-methoxybenzolthiazole)-acetonitrile (1.50 g, 7.34 mmol) using aprocedure analogous to that described in Preparation 12. The titlecompound was isolated in a yield of 1.63 g (72%). MS (m/z, ES+): 309(M+1, 60%), 465 (by-product, 100%).

Preparation 14: Synthesis of2-Benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile

The title compound was prepared from benzothiazol-2-ylacetonitrile (1.74g, 10 mmol) using a procedure analogous to that described in Preparation12. The title compound was isolated in a yield of 1.25 g (45%). MS (m/z,ES+): 279 (M+1, 100%). m/z

Preparation 15: Synthesis of Benzoxazol-2-ylacetonitrile

Compound prepared by this procedure in Sakamoto, M.; Nozaka, A.;Shimamoto, M.; Ozaki, H.; Suzuki, Y.; Yoshioka, S.; Nagano, M.; Okamura,K.; Date, T.; Tamura, O. J. Chem. Soc. Perkin Trans. I (1995), 1759 andreferences therein).

To a solution of 2-aminophenol (5.0 g, 45.8 mmol) dissolved in 115 mL ofanhydrous ethanol and 8 mL of glacial acetic acid was added malonitrile(9.08 g, 140 mmol) at 100° C. The resulting homogeneous solution wasrefluxed for 24 hours and the solvent was removed in vacuo to yield ared/brown oil. The oil was diluted with CH₂Cl₂ (100 mL) and anyremaining solids were then removed by filtration. The mother liquor waswashed twice with a saturated sodium bicarbonate solution and twice withwater, dried over magnesium sulfate, filtered and evaporated to yield5.2 g (71%) of the product as a yellow oil. This material was used inthe subsequent reaction without further purification.

Preparation 16: Synthesis of2-Benzoxazol-2-yl-3,3-bismethylsulfanylacrylonitrile

(Augustin, M.; Doelling, W.; J. Prakt. Chem. (1982) 1, 3).

To a solution of benzoxazol-2-ylacetonitrile (2.07 g, 13.1 mmol)dissolved in 35 mL of DMSO at room temperature under argon atmospherewas added carbon disulfide (1.10 g, 14.4 mmol) followed by iodomethane(5.20 g, 36.7 mmol). This solution was then cooled to 10° C. and sodiumhydride (1.05 g, 60% by wt in oil, 26.3 mmol) was added over a period ofseveral minutes. The solution was allowed to warm to room temperatureand stirred overnight. The reaction was then quenched with an ammoniumchloride solution and was extracted with 5×50 mL of ethyl acetate. Theethyl acetate was washed with brine solution (3×50 mL) and water (3×30mL). The organic phase was dried over magnesium sulfate, filtered, andevaporated to yield a red oil. The crude material was purified by flashcolumn chromatography eluting with CH₂Cl₂:MeOH=98:2 to yield 1.96 g(57%) of the desired product as a yellow oil. MS (m/z, ES+): 236.68(M+1, 100%). The following examples illustrate the preparation ofcompounds disclosed in this invention.

Example 2 Synthesis of 4-Benzothiazol-2-yl-1H-pyrazol-3-ylamine

A mixture of 2-aminobenzenethiol (100 mg, 0.8 mmol) and3-aminopyrazole-4-carbonitrile (86 mg, 0.8 mmol) in 3 g ofpolyphosphoric acid was heated at 200° C. for 3 hours. The mixture wasthen poured into ice water and was neutralized with conc. Ammoniumhydroxide solution. The resulting yellow solid was isolated byfiltration and washed with cold water to yield the title compound (70mg, 40%). MS (m/z, ES+): 217 (M+1, 100%); ¹H NMR (300 MHz, ppm,DMSO-d₆): δ 7.97 (d, ³J=7.8 Hz, 1H), 7.88 (s, 1H), 7.84 (d, ³J=8.0 Hz,1H), 7.42 (m, 1H), 7.29 (m, 1H), 6.3 (br s, 2H).

The following compounds were prepared in a manner analogous to theprocedure described in Example 2.

4-(1H-Benzoimidazol-2-yl)-1H-pyrazol-3-ylamine: The title compound(0.031 g) was prepared starting from 0.30 g (2.8 mmol) 1,2-phenyldiamineand 0.30 g (2.8 mmol) 3-aminopyrazole-4-carbonitrile. MS (m/z, ES+): 200(M+1, 100%). Yield=6%.

4-(1H-Benzoimidazol-2-yl)-1H-pyrazole-3,5-diamine: The title compound(0.047 g) was prepared starting from 0.46 g (4.22 mmol) of1,2-phenyldiamine and 0.52 g (4.22 mmol) of3,5-diaminopyrazole-4-carbonitrile. MS (m/z, ES+): 215 (M+1, 100%).Yield=5%.

2-(1H-Pyrazol-4-yl)-benzothiazole: The title compound (0.055 g) wasprepared starting from 0.11 g (0.090 mmol) of 2-aminophenol and 0.10 g(0.90 mmol) of 4-pyrazolecarboxylic acid in 5 mL of anhydrous ethanoland 1 mL of glacial acetic acid at reflux for 24 hours. MS (m/z, ES+):202 (M+1, 100%); ¹H NMR (400 MHz, ppm, DMSO-d₆) 13.45 (br s, 1H), 8.32(br s, 2H), 8.00 (dd, 2H), 7.43 (dt, 2H). Yield=34%.

Example 3 Synthesis of4-(6-Bromobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine

1. A mixture of 4-bromoaniline (1.72 g 10 mmol) and ammonium thiocyanate(3.05 g, 40 mmol) in acetic acid (15 mL) was stirred for approximately15 minutes until a homogeneous solution was obtained. A solution ofbromine (1.6 g, 10 mmol) in acetic acid (7 mL) was then added to theresulting mixture over a period of 20 minutes. The reaction mixture wasstirred overnight at room temperature. The resulting precipitate wasisolated by filtration and dried under high vacuum to afford the crudeproduct which was used in the following reaction without furtherpurification.

2. A suspension of 6-bromobenzothiazol-2-ylamine (2.29 g, 10 mmol)prepared above in sodium hydroxide (40 mL of a 6 M solution, 240 mmol)was refluxed under argon overnight. The reaction mixture was cooled inan ice bath and was acidified to between pH 3 and 5 with conc. HCl. Theresulting precipitate was isolated by filtration, washed with water anddried under high vacuum to afford the crude product that was used in thefollowing reaction without further purification.

3. A mixture of 2-amino-5-bromobenzenethiol (1.29 g, 6.3 mmol) andmalononitrile (0.66 g, 10 mmol) in ethanol (20 mL) was heated to refluxovernight. The reaction was then cooled to room temperature and theresulting precipitate was isolated by filtration to afford the crudeproduct that was used in the following reaction without furtherpurification.

4. A mixture of (6-bromobenzothiazol-2-yl)acetonitrile (4.9 mmol) andtrimethyl orthoacetate (0.71 g, 5.88 mmol) in acetic anhydride (12 mL)was heated at 100° C. for 5 h. The reaction mixture was then cooled toroom temperature. The resulting precipitate was isolated by filtrationto afford the crude product which was used in the following reactionwithout further purification.

5. A solution of 2-(6-bromobenzothiazol-2-yl)-3-methoxybut-2-enenitrile(0.96 g, 3.1 mmol) and hydrazine hydrate (0.3 mL, 5.4 mmol) in methanol(30 mL) was heated to reflux overnight. The reaction was then cooled toroom temperature. The resulting solid was isolated by filtration and waspurified by flash chromatography to yield the title product. MS (m/z,ES+): 308.9 (Br⁷⁹M+1, 100%), 310.9 (Br⁸¹ M+1, 100%). Yield=5%.

The following compounds were prepared in a manner analogous to theprocedure described in Example 3.

2-(3-Amino-5-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid amide:The title compound (23 mg) was prepared in five steps starting from 3.4g (20 mmole) of 4-aminobenzenesulfonamide. MS (m/z, ES+): 310 (M+1,100%). ¹H NMR (300 MHz, ppm, DMSO-d₆): 11.78 (br s, 1H), 8.47 (s, 1H),7.98 (d, 1H), 7.86 (d, 1H), 6.68 (br s, 2H), 7.37 (s, 2H), 2.41 (s, 3H).

4-(6-Methanesulfonylbenzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine:The title compound (46 mg) was prepared in four steps starting from 1.0g (4.4 mmol) of 2-amino-6-methanesulfonylbenzothiazol. MS (m/z, ES+):309 (M+1, 100%).

4-(6-Methoxybenzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine: The titlecompound (32 mg) was prepared in two steps starting from 140 mg (0.68mmol) of (6-methoxybenzothiazol-2-yl)acetonitrile. MS (m/z, ES+): 261(M+1, 100%).

4-(6-Fluorobenzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine: The titlecompound (55 mg) was prepared in four steps starting from 1.0 g (5.95mmol) of 2-amino-6-fluorobenzothiazole. MS (m/z, ES+): 248 (M+1, 100%).

5-Methyl-4-thiazolo[5,4-b]pyridin-2-yl-1H-pyrazol-3-ylamine: The titlecompound (162 mg) was prepared in four steps starting from 1.5 g (10mmol) of thiazolo[5,4-b]pyridin-2-ylamine. MS (m/z, ES+): 232 (M+1,100%).

4-Benzothiazol-2-yl-5-methyl-1H-pyrazol-3-ylamine: The title compound(42 mg) was prepared in two steps starting from 174 mg (1.0 mmol) ofbenzothiazol-2-ylacetonitrile. MS (m/z, ES+) 231 (M+1, 100%).

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine(474-98E): The title compound (32 mg) was prepared in two steps startingfrom 0.24 g (0.86 mmol) of(5-fluoro-6-methoxybenzothiazol-2-yl)acetonitrile. MS (m/z, ES+): 279.1(M+1, 100%).

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-carboxylic acidamide (574-3B): The title compound (64 mg) was prepared in five stepsstarting from 1.4 g (10 mmol) of 4-aminobenzamide. MS (m/z, ES+): 274.0(M+1, 100%).

N-[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-6-yl]acetamide(574-8E): The title compound (160 mg) was prepared in five stepsstarting from 1.5 g (10 mmol) of N-(4-aminophenyl)acetamide. MS (m/z,ES+): 245.1 (M−42, 100%); MS (m/z, ES−): 243.2 (M−44, 100%); ¹H NMR (300MHz, ppm, DMSO-d₆): δ 11.9-11.6 (br s, 1H), 7.79 (s, 1H), 7.73 (d, 1H),7.24 (d, 1H), 6.50 (br s, 1H), 5.78 (br s, 1H), 2.41 (s, 3H), 2.38 (brs, 3H); IR (KBr): 3518, 3207, 1607 (vs), 1544, 1506, 1023, 963, 821cm⁻¹.

4-(6-Chlorobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine (598-38):The title compound (396 mg) was prepared in four steps starting from 1.0g (5.4 mmol) of 6-chloro-2-aminobenzothiazole. During the final step, 4drops of conc HCl were added while the solution was under reflux to formthe pyrazole ring. MS (m/z, ES+): 265.0 (Cl³⁵ M+1, 100%), 267.0 (Cl³⁷M+1, 50%).

4-(4-Fluorobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine (598-45):The title compound (325 mg) was prepared in four steps starting from1.22 g (7.3 mmol) of 4-fluorobenzothiazol-2-ylamine. During the finalstep, 4 drops of conc HCl were added while the solution was under refluxto form the pyrazole ring. The solid was removed by filtration and theresulting solution was evaporated to afford the title compound. MS (m/z,ES+): 249.0 (M+1, 100%).

4-(5-Trifluoromethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine(598-66): The title compound (621 mg) was prepared in three stepsstarting from 1.0 g (4.4 mmol) of 2-amino-4-trifluoromethylbenzenethiolhydrochloride. During the final step, 4 drops of conc HCl were addedwhile the solution was under reflux to form the pyrazole ring. MS (m/z,ES+): 299.0 (M+1, 100%).

4-(7-Chloro-4-methoxybenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine(598-58): The title compound (176 mg) was prepared in four stepsstarting from 2.1 g (9.8 mmol) of7-chloro-4-methoxybenzothiazol-2-ylamine. During the final step, acatalytic amount of p-toluenesulfonic acid was added while the solutionwas under reflux to form the pyrazole ring. MS (m/z, ES+): 295.0(Cl³⁵M+1, 100%), 297.0 (Cl³⁷M+1, 50%).

2-(3-Amino-5-methyl-1H-pyrazol-4-yl)benzothiazole-6-carboxylic acid(574-26E1): The title compound (1.58 g) was prepared in five stepsstarting from 16.5 g (100 mmol) of 4-aminobenzoic acid ethyl ester.During the final step, 4 drops of conc. HCl were added while thesolution was under reflux to form the pyrazole ring. MS (m/z, ES+): 275(M+1, 100%).

4-(6-Bromo-5-fluorobenzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine(574-28E): The title compound (765 mg) was prepared in five stepsstarting from 5.0 g (26.3 mmol) of 4-bromo-3-fluoroaniline. During thefinal step, a catalytic amount of p-toluenesulfonic acid was added whilethe solution was under reflux to form the pyrazole ring. MS (m/z, ES+):327.0 (Br⁷⁹M+1, 100%), 329.0 (Br⁸¹ M+1, 100%).

2-(3-Amino-5-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(2,6-dimethyl-pyrimidin-4-yl)-amide (474-92B): The title compound (93mg) was prepared in five steps starting from sulfisomidine with theexception that in step 2 KOH was replace with sodium sulfide (5.0 g). MS(m/z, ES+): 416.0 (M+1, 100%).

2-(3-Amino-5-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acidmethylamide (503-55B): The title compound (168 mg) was prepared in fivesteps starting from 2.0 g (11 mmole) of4-amino-N-methylbenzenesulfonamide. MS (m/z, ES+): 324.0 (M+1, 100%). ¹HNMR (400 MHz, ppm, DMSO-d₆): δ 11.80-12.15 (br, 1H), 8.46 (s, 1H),7.99-5.87 (br m, 2H), 7.79 (d, 1H), 7.74 (dd, 1H), 6.72 (br s, 2H), 2.43(s, 3H), 2.42 (s, 3H).

2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-6-sulfonicacid methylamide (574-37E): The title compound (3 mg) was prepared infive steps starting from 1.9 g (10 mmol) of4-amino-2-fluoro-N-methylbenzenesulfonamide. MS (m/z, ES+): 342.2 (M+1,100%).

4-(5-Fluoro-6-methylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine(610-89): The title compound (24 mg) was prepared in five steps startingfrom 3-fluoro-4-methylaniline. MS (m/z, ES+): 263.04 (M+1, 100%).

5-Methyl-4-(4,5,6-trifluorobenzothiazol-2-yl)-1H-pyrazol-3-ylamine(598-75): The title compound (520 mg) was prepared in five stepsstarting from 3.0 g (20 mmol) of 2,3,4-trifluoroaniline. During thefinal step, 4 drops of conc. HCl were added while the solution was underreflux to form the pyrazole ring. MS (m/z, ES+): 285.0 (M+1, 100%).

2-(3-Amino-5-methyl-1H-pyrazol-4-yl)benzothiazole-7-carboxylic acidmethyl ester (598-85): The title compound (33 mg) was prepared in twosteps starting from 1.1 g (5.6 mmol) 2-aminobenzothiazole-7-carboxylicacid. Excess trimethyl orthoacetate (8.0 mmol) was used to convert theacid group to the methyl ester. During the final step, a catalyticamount of p-toluenesulfonic acid was added while the solution was underreflux to form the pyrazole ring. MS (m/z, ES+): 289.2 (M+1, 100%).

2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-6-carboxylicacid methyl ester (574-45F): The title compound (50 mg) was prepared infive steps starting from 860 mg (5.5 mmol) of 4-amino-2-fluorobenzoicacid. Excess trimethyl orthoacetate was used to convert the acid groupto a methyl ester during step 4. During the final step, 4 drops of conc.HCl were added while the solution was under reflux to form the pyrazolering. MS (m/z, ES+): 307.0 (M+1, 100%).

4-(5-Trifluoromethylbenzothiazol-2-yl)-1H-pyrazol-3-ylamine: The titlecompound (29 mg) was prepared in three steps starting from 1.1 g (5.0mmol) of 2-amino-4-trifluoromethylbenzenethiol hydrochloride. Triethylorthorcarbonate (1.2 equiv.) was substituted for trimethyl orthoacetate.MS (m/z, ES+): 285 (M+1, 100%).

4-(6-Fluorobenzothiazol-2-yl)-1H-pyrazol-3-ylamine (474-67B): The titlecompound (37 mg) was prepared in four steps starting from2-amino-6-fluorobenzathiazole. Triethyl orthorcarbonate (1.2 equiv.) wassubstituted for trimethyl orthoacetate. MS (m/z, ES+): 235.0 (M+1,100%).

2-(3-Amino-1H-pyrazol-4-yl)-5-fluorobenzothiazole-6-sulfonic acid amide(574-42B): The title compound (58 mg) was prepared in five stepsstarting from 4-amino-2-fluoro-N-methyl-benzenesulfonamide. Triethylorthorcarbonate (1.2 equiv.) was substituted for trimethyl orthoacetate.MS (m/z, ES+): 314.0 (M+1, 100%); ¹H NMR (300 MHz, ppm, DMSO-d₆): δ12.16 (br s, 1H), 8.46 (d, 7.1 Hz, 1H), 7.82 (d, 1H), 7.8-8.0 (m, 1H),7.63 (s, 2H), 6.7-5.5 (2H).

4-(5-Fluoro-6-methylbenzothiazol-2-yl)-2H-pyrazol-3-ylamine (610-93):The title compound (54 mg) was prepared in five steps starting from3-fluoro-4-methylaniline. Triethyl orthorcarbonate (1.2 equiv.) wassubstituted for trimethyl orthoacetate. MS (m/z, ES+): 249.05 (M+1,100%).

2-(5-Amino-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid methylamide(599-77B2): The title compound (107 mg) was prepared in five stepsstarting from 4-amino-N-methylbenzenesulfonamide. N,N-Dimethylformamidedimethyl acetal (1.2 equiv.) was substituted for trimethyl orthoacetate.MS (m/z, ES+): 310.0 (M+1, 100%); ¹H NMR (300 MHz, ppm, DMSO-d₆): δ 12.1(br s, 1H), 8.43 (d, 1H), 8.0 (br s, 1H), 7.97 (d, 1H), 7.78 (dd, 1H),7.38 (q, 1H), 6.5 (br s, 1H), 5.9 (br s, 1H), 2.44 (d, 3H).

4-Benzothiazol-2-yl-5-ethyl-1H-pyrazol-3-ylamine: The title compound (75mg) was prepared in two steps starting from 174 mg (1.0 mmol) ofbenzothiazol-2-ylacetonitrile and 211 mg (1.2 mmol) of triethylorthopropionate. MS (m/z, ES+): 245 (M+1, 100%).

Example 4 Synthesis of2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-5-sulfonic acidmethylamide (574-12E)

1. To a solution of 4-chloro-3-nitrobenzenesulfonyl chloride (2.0 g, 7.8mmol) in THF (10 mL) was added excess methylamine (2M solution in THF).The reaction solution was stirred at room temperature for 1 hr. Waterwas then added and the product was extracted into chloroform. Thecombined extracts were washed, dried over sodium sulfate, filtered andevaporation to yield 2.3 g of the crude4-chloro-N-methyl-3-nitrobenzenesulfonamide as an oil. The crudematerial was used in the subsequent step without further purification.

2. An ethanolic solutions of sodium disulphide was prepared bydissolving sodium sulfide nonahydrate (2.0 g, 8.5 mmol) in hot ethanol(9 mL) and then adding sulfur (0.27 g, 8.5 mmol). This was cooled toroom temperature and added dropwise to a solution of the above preparedcrude 4-chloro-N-methyl-3-nitrobenzenesulfonamide (2.3 g) in ethanol (15mL). After the addition had been completed, the resulting precipitatewas isolated by filtration and was washed with ethanol to yield 1.5 g(79% for two steps) of4,4′-dithiobis(N-methyl-3-nitrobenzenesulfonamide) as a yellow powder.MS (m/z, ES−): 492.9 (M−1, 100%).

3. To a solution of 4,4′-dithiobis(N-methyl-3-nitrobenzenesulfonamide)(1.5 g, 3.1 mmol) in ethanol (70 mL) was added a solution of tinchloride dihydrate (5.5 g) in hydrochloric acid (10 mL of a 2Nsolution). The reaction mixture was heated to reflux over night and thenmalononitrile (660 mg, 10 mmol) was added. The resulting mixture wasrefluxed for an additional 5 hrs and was then cooled to room temperatureand diluted with water. The solid was isolated by filtration and waspurified by flash column chromatography to yield 120 mg (8%) of2-cyanomethylbenzothiazole-5-sulfonic acid methylamide.

4. A mixture of 2-cyanomethylbenzothiazole-5-sulfonic acid methylamide(120 mg, 0.45 mmol) and trimethyl orthoracetate (270 mg, 2.2 mmol) inacetic anhydride (2 mL) was heated at 100° C. for 5 hrs. The reactionmixture was cooled to room temperature. The resulting precipitate wasisolated by filtration to yield 120 mg (83%) of2-(1-cyano-2-methoxypropenyl)benzothiazole-5-sulfonic acid methylamide.The product was used in the subsequent step without furtherpurification. MS (m/z, ES+): 324 (M+1, 100%).

5. To a suspension of2-(1-cyano-2-methoxypropenyl)benzothiazole-5-sulfonic acid methylamide(120 mg, 0.37 mmol) in 4 mL of methanol, was added hydrazine hydrate (20μL). The mixture was heated to reflux for 5 hrs and then 1 drop of conc.hydrochloric acid was added to the reaction mixture. The mixture wasrefluxed for an additional 5 min and was then allowed to cool. Thevolume of solvent was reduced under reduced pressure and a solid formed.The solid was isolated by filtration and was washed with methanol andthen water and dried to yield 51 mg (43%) of the title compound as acream coloured powder. MS (m/z, ES+): 324.0 (M+1, 100%).

The following compounds were prepared in a manner analogous to theprocedure described in Example 4.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-5-carboxylic acidethyl ester (574-19): The title compound (0.50 g) was prepared in foursteps starting from 5.0 g (22 mmol) of 4-chloro-3-nitrobenzoic acidethyl ester. MS (m/z, ES+): 303 (M+1, 100%).

Example 5 Synthesis of4-(5-Fluorobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine

1. To a solution of 4-fluoro-2-nitroaniline (1.6 g, 10 mmol) in conc.sulfuric acid (3 mL) and water (3 mL) at 5° C. was added a solution ofsodium nitrite (760 mg, 11 mmol) in 3 mL of water. After stirring for 40minutes, a solution of potassium thiocyanate (1.0 g, 10 mmol) in 2 mL ofwater was added. The solution was then poured into a vigorously stirringsuspension of cuprous thiocyanate (1.8 g, 15 mmol) in 6 mL of water at5° C. The resulting mixture was stirred for 2 hrs and left to stand inthe refrigerator overnight. The resulting solid was isolated byfiltration and was washed with hot dichloromethane. The dichloromethaneextracts were dried with sodium sulfate, filtered and evaporated toyield 1.90 g (96%) of a yellow solid which was used in the subsequentreaction without further purification. IR (KBr disc): 2158 (m) cm⁻¹.

2. To a suspension of the crude product prepared above (1.90 g) inethanol (10 mL) was added a solution of sodium sulfide in water (30 mL).The resulting deep red mixture was heated to reflux for 1 hr. Themixture was cooled, acidified to approximately pH 5 and extracted withdichloromethane. The organic extracts were combined, washed with water,dried over sodium sulfate, filtered, and evaporated to yield 2.1 g ofcrude 4-fluoro-2-nitrobenzenethiol which was used in the subsequent stepwithout further purification. MS (m/z, ES+): 174.0 (M+1, 100%).

3. To a solution of 2.1 g of the crude 4-fluoro-2-nitrobenzenethiolprepared above in ethanol was added SnCl₂ dihydrate (6.7 g, ca. 3equi.). The mixture was heated to reflux overnight. To this mixture wasadded molanonitrile (400 mg). After refluxing for an additional 5 hrs,the reaction mixture was cooled and filtered. The volume of solvent wasreduced by evaporation and water was added to induce the formation of aprecipitate. The crude product (940 mg) was isolated by filtration andwas then purified by flash chromatography eluting with hexanes:EtOAc=1:1to yield 360 mg (19% for 3 steps) of5-fluorobenzothiazol-2-yl)acetonitrile as a pale greenish solid. MS(m/z, ES+): 193 (M+1, 100%).

4. A mixture of 5-fluorobenzothiazol-2-yl)acetonitrile (360 mg, 1.9mmol) and trimethyl orthoracetate (270 mg, 2.3 mmol) in acetic anhydride(2 mL) was heated at 100° C. for 5 hrs. The reaction mixture was cooledand the resulting precipitate was isolated by filtration, washed withether, and dried to yield 330 mg (71%) of2-(5-fluorobenzothiazol-2-yl)-3-methoxybut-2-enenitrile as a red solid.MS (m/z, ES+): 249 (M+1, 100%).

5. A suspension of2-(5-fluorobenzothiazol-2-yl)-3-methoxybut-2-enenitrile (330 mg, 1.3mmol) and hydrazine hydrate (70 μL) in methanol (40 mL) was heated toreflux overnight. The solution was then cooled to room temperature andthe resulting solid was isolated by filtration. The crude material wasrecrystallized from ethanol to yield 260 mg (78%, 10% for five steps) ofthe title compound. MS (m/z, ES+): 249.0 (M+1, 100%).

The following compounds were prepared in a manner analogous to theprocedure described in Example 5.

4-(5-Methoxy-benzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine (574-7E):The title compound (16 mg) was prepared in five steps starting from 1.6g (7.6 mmol) of 4-methoxy-2-nitroaniline. MS (m/z, ES+): 261.0 (M+1,100%).

Example 6 Synthesis of4-Benzothiazol-2-yl-5-methylsulfanyl-1H-pyrazol-3-ylamine

To a suspension of2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile (50 mg, 0.18mmol) in ethanol (2 mL) was added hydrazine hydrate (15 μL). The mixturewas heated to reflux for 2 hours. The reaction mixture was then cooledand water was added. The resulting precipitate was isolated byfiltration to yield 21 mg (44%) of the title compound as a yellow solid.MS (m/z, ES+): 263 (M+1, 100%).

Example 7 Synthesis of 4-Benzothiazol-2-yl-1H-pyrazole-3,5-diamine

To a suspension of2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile (140 mg, 0.5mmol) in ethanol (5 mL) was added a solution of ammonia (6 mL of a 2 Nsolution in ethanol, 12 mmol). The reaction was heated to 70° C. for 5hours in a sealed reaction vessel. After cooling to room temperature,hydrazine hydrate (60 μL, 1.7 mmol) was added and the reaction washeated to reflux for 2 days. The solvent was then evaporated and thecrude material was purified by flash chromatography eluting withCHCl₃:MeOH=9:1 to yield 0.027 g (23%) of the title compound as a palepink powder. MS (m/z, ES+): 232 (M+1, 100%). ¹H NMR (ppm, 300 MHz,DMSO-d₆): 810.85 (br s, 1H), 7.98 (d, ³J=7.8 Hz, 1H), 7.78 (d, ³J=8.0Hz, 1H), 7.39 (dd, ³J=7.8 Hz, ³J=8.0 Hz, 1H), 7.23 (dd, ³J=7.8 Hz,³J=8.0 Hz, 1H), 5.55 (br s, 4H).

Example 8 Synthesis of 4-Benzoxazol-2-yl-1H-pyrazole-3,5-diamine

The title compound (0.020 g) was prepared according to the procedure asdescribed in Example 7 starting from 1.90 g (7.3 mmol) of2-benzoxazol-2-yl-3,3-bismethylsulfanylacrylonitrile with the followingmodifications. A 300 mL of a saturated ammonium solution in ethanol wasused and the reaction was refluxed for 1 hr prior to the evaporation ofthe solvent and purification of the resulting crude material. Thisintermediate was then treated with hydrazine hydrate to afford theproduct. MS (m/z, ES+): 216.72 (M+1, 100%). Yield=1%.

Example 9 Synthesis of4-Benzothiazol-2-yl-N⁵-benzyl-1H-pyrazole-3,5-diamine

A solution of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile(200 mg, 0.73 mmol) and benzylamine (160 mg, 1.5 mmol) in 50 mL ofethanol was heated to reflux for 90 minutes. Hydrazine hydrate (35 μL,1.0 mmol) was then added to the reaction mixture. The solution washeated to reflux until the reaction was complete as determined by TLCanalysis. The reaction solution was allowed to cool to room temperatureand the title compound (124 mg) was isolated by filtration, washed withethanol and dried under high vacuum. MS (m/z, ES+): 322 (M+1, 100%); ¹HNMR (300 MHz, ppm, DMSO-d₆): δμ11.32 (br s, 1/3H), 10.89 (s, br., 2/3H),7.98 (d, ³J=7.7 Hz, 1H), 7.82 (d, ³J=8.0 Hz, 1H), 7.41-7.28 (m, 6H),7.22 (m, 1H), 6.15 (br s, 9/10H), 4.95 (br s, 1/10H), 4.48 (d, ³J=4.7Hz, 1H). Yield=53%.

The following compounds were prepared in a manner analogous to theprocedure described in Example 9.

4-Benzothiazol-2-yl-5-pyrrolidin-1-yl-1H-pyrazol-3-ylamine: The titlecompound (53 mg) was prepared in two steps starting from 140 mg (0.50mmol) of 2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 53mg (0.75 mmol) of pyrrolidine. MS (m/z, ES+): 286 (M+1, 100%).Yield=37%.

4-Benzothiazol-2-yl-N⁵-ethyl-1H-pyrazole-3,5-diamine (48 mg) wasprepared in two steps starting from 139 mg (0.50 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 0.059 mL ofa 70% w/w solution (0.75 mmol) of ethylamine in water. MS (m/z, ES+):260 (M+1, 100%). Yield=37%.

4-Benzothiazol-2-yl-5-morpholin-4-yl-1H-pyrazol-3-ylamine: The titlecompound (46 mg) was prepared in two steps starting from 139 mg (0.50mmol) of 2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 62mg (0.75 mmol) of morpholine. MS (m/z, ES+): 302 (M+1, 100%). Yield=30%.

4-Benzothiazol-2-yl-5-(4-methylpiperazin-1-yl)-1H-pyrazol-3-ylamine: Thetitle compound (7 mg) was prepared in two steps starting from 139 mg(0.50 mmol) of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrileand 75 mg (0.75 mmol) of 1-methylpiperazine. MS (m/z, ES+): 315 (M+1,100%). Yield=4%.

4-Benzothiazol-2-yl-N⁵-(3,5-dichlorophenyl)-1H-pyrazole-3,5-diamine: Thetitle compound (30 mg) was prepared in two steps starting from 100 mg(0.36 mmol) of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrileand 117 mg (0.72 mmol) of 3,5-dichlorophenylamine. MS (m/z, ES+): 376(Cl³⁵Cl³⁵M+1, 100%), 378 (Cl³⁷Cl³⁵M+1, 70%). Yield=22%.

4-Benzothiazol-2-yl-N⁵-(3-trifluoromethanesulfonyl-phenyl)-1H-pyrazole-3,5-diamine:The title compound (9 mg) was prepared in two steps starting from 100 mg(0.36 mmol) of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile,81 mg (0.72 mmol) of 3-trifluoromethane-sulfonylphenylamine and 0.04 mLof triethylamine. MS (m/z, ES+): 440 (M+1, 100%). Yield=6%.

4-(5-Amino-4-benzothiazol-2-yl-2H-pyrazol-3-ylamino)-N-thiazol-2-yl-benzenesulfonamide:The title compound (20 mg) was prepared in two steps starting from 100mg (0.36 mmol) of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrileand 93 mg (0.36 mmol) of 4-amino-N-thiazol-2-ylbenzenesulfonamide. MS(m/z, ES+): 470 (M+1, 100%). Yield=12%.

4-Benzothiazol-2-yl-N⁵-quinolin-6-yl-1H-pyrazole-3,5-diamine: The titlecompound (91 mg) was prepared in two steps starting from 100 mg (0.36mmol) of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile and 103mg (0.72 mmol) of 6-aminoquinoline. MS (m/z, ES+): 359 (M+1, 100%).Yield=70%.

4-Benzothiazol-2-yl-N³-quinolin-5-yl-1H-pyrazole-3,5-diamine: The titlecompound (33 mg) was prepared in two steps starting from 100 mg (0.36mmol) of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile and 103mg (0.72 mmol) of 5-aminoquinoline. MS (m/z, ES+): 359 (M+1, 100%).Yield=25%.

4-Benzothiazol-2-yl-N⁵-pyridin-3-yl-1H-pyrazole-3,5-diamine: The titlecompound (69 mg) was prepared in two steps starting from 100 mg (0.36mmol) of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile and 68mg (0.72 mmol) of 3-aminopyridine. MS (m/z, ES+): 309 (M+1, 100%).Yield=62%.

trans-2-(5-Amino-4-benzothiazol-2-yl-2H-pyrazol-3-ylamino)cyclopentanol:The title compound (69 mg) was prepared in two steps starting from 139mg (0.50 mmol) of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrileand 75 mg (0.75 mmol) of trans-2-amino-cyclopentanol. MS (m/z, ES+): 316(M+1, 100%). Yield=44%.

4-Benzothiazol-2-yl-N⁵-pyridin-4-ylmethyl-1H-pyrazole-3,5-diamine: Thetitle compound (32 mg) was prepared in two steps starting from 83 mg(0.30 mmol) of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrileand 64 mg (0.60 mmol) of 4-(aminomethyl)pyridine. MS (m/z, ES+): 323(M+1, 100%). Yield=33%.

4-Benzothiazol-2-yl-N⁵-pyridin-3-ylmethyl-1H-pyrazole-3,5-diamine(474-42B): The title compound (33 mg) was prepared in two steps startingfrom 83 mg (0.30 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 64 mg (0.60mmol) of 3-(aminomethyl)pyridine. MS (m/z, ES+): 323.1 (M+1, 100%).Yield=34%.

4-Benzothiazol-2-yl-N⁵-(2-morpholin-4-ylethyl)-1H-pyrazole-3,5-diamine(474-42C): The title compound (40 mg) was prepared in two steps startingfrom 83 mg (0.30 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 78 mg (0.60mmol) of 2-morpholin-4-ylethylamine. MS (m/z, ES+): 345.2 (M+1, 100%).Yield=43%.

4-Benzothiazol-2-yl-N³-(3-imidazol-1-ylpropyl)-1H-pyrazole-3,5-diamine(590-13-2): The title compound (69 mg) was prepared in two stepsstarting from 280 mg (1.0 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 260 mg (2.1mmol) of histamine. MS (m/z, ES+): 340.1 (M+1, 65%), 272 (M−67, 100%).Yield=43%.

4-Benzothiazol-2-yl-N³-(3-dimethylaminopropyl)-1H-pyrazole-3,5-diamine(590-14): The title compound (130 mg) was prepared in two steps startingfrom 140 mg (0.50 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 82 mg (0.80mmol) of N,N-dimethylaminopropylamine. MS (m/z, ES+): 317.1 (M+1, 100%).Yield=80%.

4-Benzothiazol-2-yl-N³-(2-pyrrolidin-1-ylethyl)-1H-pyrazole-3,5-diamine(590-18): The title compound (126 mg) was prepared in two steps startingfrom 140 mg (0.50 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 62 mg (0.55mmol) of 1-(2-aminoethyl)pyrrolidine. MS (m/z, ES+): 329.1 (M+1, 100%),258.1 (M-C₄H₈N, 40%). Yield=88%.

4-Benzothiazol-2-yl-N³-(2-methoxyethyl)-1H-pyrazole-3,5-diamine(590-27): The title compound (10 mg) was prepared in two steps startingfrom 100 mg (0.36 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 30 mg (0.40mmol) of 2-methoxyethylamine. MS (m/z, ES+): 290.1 (M+1, 100%).Yield=10%.

3-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)propan-1-ol(590-28): The title compound (83 mg) was prepared in two steps startingfrom 100 mg (0.36 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 30 mg (0.40mmol) of 3-propanolamine. MS (m/z, ES+): 290.1 (M+1, 100%). Yield=80%.

4-[(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-methyl]benzenesulfonamide(590-29): The title compound (35 mg) was prepared in two steps startingfrom 100 mg (0.37 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile, 93 mg (0.42mmol) of 4-(aminomethyl)benzenesulfonamide hydrochloride hydrate, and100 mg (1.0 mmol) of triethylamine. MS (m/z, ES+): 401.1 (M+1, 100%).Yield=24%.

N-[2-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-ethyl]acetamide(590-33): The title compound (93 mg) was prepared in two steps startingfrom 140 mg (0.50 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 93 mg (0.55mmol) of N-acetylethylenediamine. MS (m/z, ES+): 317.1 (M+1, 99%), 299.1(M−17, 100%). Yield=59%.

4-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)butan-1-ol (590-44):The title compound (54 mg) was prepared in two steps starting from 100mg (0.36 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 40 mg (0.45mmol) of 4-amino-1-butanol. MS (m/z, ES+): 304.1 (M+1, 100%). Yield=50%.

4-Benzothiazol-2-yl-5-piperazin-1-yl-2H-pyrazol-3-ylamine (590-58): Thetitle compound (50 mg) was prepared in two steps starting from 75 mg(0.30 mmol) of 2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrileand 28 mg (0.36 mmol) of piperazine. MS (m/z, ES+): 301.1 (M+1, 100%).Yield=55%.

4-Benzothiazol-2-yl-N³-piperidin-4-ylmethyl-1H-pyrazole-3,5-diamine(610-38E): The title compound (14 mg) was prepared in two steps startingfrom 100 mg (0.36 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 47 mg (0.41mmol) of 4-aminomethylpiperidine. MS (m/z, ES+): 329.2 (M+1, 25%), 244.1(M−84, 45%), 232.1 (M−96, 100%). Yield=12%.

4-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)butyric acid(523-29-B): The title compound (50 mg) was prepared in two stepsstarting from 530 mg (2.0 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile, 250 mg (2.3mmol) of 4-aminobutyric acid and 0.6 mL of triethylamine. MS (m/z, ES+):318.1 (M+1, 100%), 300.1 (M-H2O, 95%); ¹H NMR (300 MHz, ppm, DMSO-d₆): δ11.5 (br s, 2H), 7.8 (d, 1H), 7.7 (d, 1H), 7.4 (d, 1H), 7.2 (d, 1H), 6.0(br s, 1H), 5.5 (br s, 2H), 3.2 (m, 2H), 2.3 (t, 2H), 1.8 (m, 2H).Yield=8%.

4-Benzothiazol-2-yl-N-3-[2-(1H-imidazol-4-yl)-ethyl]-1H-pyrazole-3,5-diamine(523-31C): The title compound (310 mg) was prepared in two stepsstarting from 530 mg (2.0 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile, 500 mg (4.5mmol) of histamine and 0.6 mL of triethylamine. MS (m/z, ES+): 326.09(M+1, 100%). Yield=48%.

2-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-ethanol (523-34A):The title compound (140 mg) was prepared in two steps starting from 530mg (2.0 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile, 500 mg (8.0mmol) of monoethanolamine and 0.5 mL of triethylamine. MS (m/z, ES+):276.02 (100%, M+1). Yield=25%.

4-[2-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-ethyl]-phenol(523-36): The title compound (210 mg) was prepared in two steps startingfrom 530 mg (2.0 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile, 0.60 g (4.4mmol) of 4-(2-aminoethyl)-phenol and 0.6 mL of triethylamine. MS (m/z,ES+): 352.05 (M+1, 100%). Yield=30%.

4-Benzothiazol-2-yl-N³-(3-methylbutyl)-1H-pyrazole-3,5-diamine(523-27A): The title compound (150 mg) was prepared in two stepsstarting from 530 mg (2.0 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile, 220 mg (3.0mmol) of N,N-dimethylaminoethylendiamine and 0.6 mL of triethylamine. MS(m/z, ES+): 303.2 (M+1, 100%). Yield=25%.

4-Benzothiazol-2-yl-N³-[2-(1H-indol-3-yl)-ethyl]-1H-pyrazole-3,5-diamine(590-37): The title compound (45 mg) was prepared in two steps startingfrom 100 mg (0.36 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 62 mg (0.39mmol) of tryptamine. MS (m/z, ES+): 375.1 (M+1, 100%). Yield=49%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-N³-[2-(3H-imidazol-4-yl)-ethyl]-1H-pyrazole-3,5-diamine(597-33A): The title compound (35 mg) was prepared in two steps startingfrom 80 mg (0.25 mmol) of2-(5-fluoro-6-methoxybenzothiazole)-3,3-bis-methylsulfanylacrylonitrileand 30 mg (0.27 mmol) of histamine. MS (m/z, ES+): 374.1 (M+1, 100%).Yield=35%.

N-{2-[5-Amino-4-(5-fluoro-6-methoxybenzothiazole)-1H-pyrazol-3-ylamino]-ethyl}acetamide(597-35): The title compound (200 mg) was prepared in two steps startingfrom 350 mg (1.1 mmol) of2-(5-fluoro-6-methoxybenzothiazole)-3,3-bis-methylsulfanylacrylonitrileand 140 μL, (1.3 mmol) of N-acetylethylenediamine. MS (m/z, ES+): 365.1(M+1, 100%). Yield=51%.

4-(5-Fluoro-6-methoxybenzothiazole)-5-piperizin-1-yl-2H-pyrazol-3-ylamine(597-36): The title compound (20 mg) was prepared in two steps startingfrom 120 mg (0.37 mmol) of2-(5-fluoro-6-methoxybenzothiazole)-3,3-bis-methylsulfanylacrylonitrileand 40 mg (0.46 mmol) of piperizine. MS (m/z, ES+): 349.1 (M+1, 100%).Yield=16%.

N³-(2-Dimethylaminoethyl)-4-(5-fluoro-6-methoxybenzothiazole)-1H-pyrazole-3,5-diamine(597-40A): The title compound (35 mg) was prepared in two steps startingfrom 100 mg (0.31 mmol) of2-(5-fluoro-6-methoxybenzothiazole)-3,3-bis-methylsulfanylacrylonitrileand 40 μL (0.36 mmol) of N,N-dimethylethylenediamine. MS (m/z, ES+):351.1 (M+1, 100%). Yield=33%.

N³-(3-Dimethylaminopropyl)-4-(5-fluoro-6-methoxybenzothiazole)-1H-pyrazole-3,5-diamine(597-40B): The title compound (40 mg) was prepared in two steps startingfrom 100 mg (0.31 mmol) of2-(5-fluoro-6-methoxybenzothiazole)-3,3-bis-methylsulfanylacrylonitrileand 46 μL (0.37 mmol) of 3-(dimethylamino)propylamine. MS (m/z, ES+):365.1 (M+1, 100%). Yield=35%.

3-[5-Amino-4-(5-fluoro-6-methoxybenzothiazol-2-yl)-1H-pyrazol-3-ylamino]-propanol(597-45): The title compound (72 mg) was prepared in two steps startingfrom 100 mg (0.31 mmol) of2-(5-fluoro-6-methoxybenzothiazole)-3,3-bis-methylsulfanylacrylonitrileand 30 μL (0.39 mmol) of 3-aminopropanol. MS (m/z, ES+): 338.3 (M+1,100%). Yield=70%.

N³-[2-(3H-Imidazol-4-yl)-ethyl]-4-(6-methoxybenzothiazol-2-yl)-1H-pyrazole-3,5-diamine(597-42): The title compound (72 mg) was prepared in two steps startingfrom 100 mg (0.32 mmol) of2-(6-methoxybenzothiazole)-3,3-bis-methylsulfanylacrylonitrile and 36 mg(0.32 mmol) of histamine. MS (m/z, ES+): 356.1 (M+1, 100%). Yield=28%.

4-(6-Methoxybenzothiazole)-5-piperizin-1-yl-2H-pyrazol-3-ylamine(597-43): The title compound (39 mg) was prepared in two steps startingfrom 100 mg (0.32 mmol) of2-(6-methoxybenzothiazole)-3,3-bis-methylsulfanylacrylonitrile and 28 mg(0.33 mmol) of piperizine. MS (m/z, ES+): 331.1 (M+1, 100%). Yield=36%.

Example 10 Synthesis ofN³-(4-Amino-phenyl)-4-benzothiazol-2-yl-1H-pyrazole-3,5-diamine (590-24)

1. A mixture of 2-benzothiazol-2-yl-3,3-bis-methylsulfanyl-acrylonitrile(140 mg, 0.50 mmol) and 4′-acetylaminoaniline (90 mg, 0.60 mmol) inethanol (5 mL) was refluxed for 3 hrs. A yellow solid formed uponcooling of the reaction mixture. The resulting precipitate was isolatedby filtration and was washed with ethanol to yield 150 mg (78%) ofN-[4-(2-benzothiazol-2-yl-2-cyano-1-methylsulfanylvinylamino)phenyl]acetamide.MS (m/z, ES+): 381.1 (M+1, 100%).

2. The product from the above reaction (150 mg, 0.39 mmol) and hydrazinehydrate (50 mg, 1.0 mmol) in ethanol (5 mL) were heated to refluxovernight. Upon evaporation of approximately half of the solvent, asolid formed. The solid was isolated by filtration and was washed withethanol to yield 33 mg (23%) ofN-[4-(5-amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)phenyl]acetamide.MS (m/z, ES+): 365.1 (M+1, 100%). The mother liquor was concentrated toyield an additional 110 mg of the crude product as a red solid.

3. The crude product isolated above (110 mg, 0.3 mmol) was refluxed in amixture of ethanol (10 mL) and concentrated HCl (5 mL) for 1 hr. Ayellow solid formed upon cooling. The solid was isolated by filtrationand was washed with ethanol. The resulting hydrochloride salt (51 mg)was dissolved in water (10 mL) and adjusted to neutral pH by theaddition of dilute NaOH solution. The product was extracted into ethylacetate, the combined extracts were washed with brine, dried overanhydrous sodium sulfate, filtered and evaporated to yield 26 mg (27%)of the title compound as a cream coloured solid. MS (m/z, ES+): 323.1(M+1, 100%).

The following compounds were prepared in a manner analogous to theprocedure described in Example 10.

N³-(2-Aminoethyl)-4-benzothiazol-2-yl-1H-pyrazole-3,5-diamine (590-46):The title compound (43 mg) was prepared in three steps starting from 140mg (0.50 mmol) of2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile and 65 mg (0.55mmol) of N-acetylethylenediamine. MS (m/z, ES+): 275.1 (M+1, 100%).Yield=54%.

Example 114-Benzothiazol-2-yl-N³-(2-ethylaminoethyl)-1H-pyrazole-3,5-diamine(590-73)

1. A mixture of 2-benzothiazol-2-yl-3,3-bis-methylsulfanylacrylonitrile(200 mg, 0.74 mmol) and N-acetylethylenediamine (83 mg, 0.81 mmol) inethanol (10 mL) was refluxed for 90 min. To this solution was addedhydrazine hydrate (61 mg, 1.2 mmol), and the resulting mixture wasrefluxed overnight. Solids formed upon cooling. The resulting crystalswere filtered and washed with ethanol to yield 150 mg (66%) ofN-[2-(5-amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)ethyl]acetamide.MS (ES+): 317.1 (M+1, 100%).

2.N-[2-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)ethyl]acetamide(80 mg, 0.25 mmol) and lithium aluminum hydride (92 mg) were refluxed inanhydrous THF (10 mL) for 3 hrs. The resulting mixture was poured into asaturated ammonium chloride solution (50 mL). The aqueous phase wasextracted with ethyl acetate (3×50 mL). The combined extracts werewashed with brine, dried over anhydrous sodium sulfate, filtered andevaporated to yield 54 mg of crude product. The crude product waspurified by flash column chromatography eluting with CH₂Cl₂:MeOH=5:1with 0.5% NH₃. The title compound was isolated in a yield of 36 mg(48%). MS (m/z, ES+): 303.1 (M+1, 100%).

Example 12 Synthesis of2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid(2-hydroxyethyl)-amide (474-86D)

1. The starting material4-benzothiazol-2-yl-5-methyl-2H-pyrazol-3-ylamine (500 mg, 2.2 mmol) wasslowly added to neat chlorosulfonic acid (2.5 mL) which had been cooledin an ice bath. The solution was heated at 150° C. for 5 hrs. Thereaction mixture was then poured over ice and the resulting solids wereisolated by filtration. The solids were dried to yield 770 mg of thecrude product as a 3:1 mixture of2-(5-amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid and2-(5-amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-4-sulfonyl chloride.The crude product was used in the next step without furtherpurification.

2. The crude product (130 mg, 0.39 mmol) from the preparation of2-(5-amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonyl chloridewas suspended in chloroform. Triethylamine (0.1 mL) and 2-aminoethanol(26 mg, 0.43 mmol) were then added. The reaction mixture was stirred atroom temperature overnight. The solvent was evaporated and the productwas purified using preparative TLC eluting withEtOAc:hexanes:MeOH=6/4/0.3 to yield 22 mg (16%) of the product as acream coloured powder. MS (m/z, ES+): 354 (M+1, 100%); ¹H NMR (400 MHz,ppm, DMSO-d₆): δ 12.25-11.75 (br s, 1H, exchangeable), 8.47 (s, 1H),7.99 (d, J=8.5 Hz, 1H), 7.81 (d, J=8.5 Hz, 1H), 7.59 (br s, 1H, SO₂NH,exchangeable), 7-5.5 (br s, 2H, exchangeable), 4.66 (t, J=5.6 Hz, 1H,OH, exchangeable), 3.34 (m, J=6.2 Hz, 2H), 2.80 (br s, 2H), 2.41 (s,3H).

The following compounds were prepared in a manner analogous to theprocedure described in Example 12.

5-Methyl-4-[6-(4-methylpiperazine-1-sulfonyl)-benzothiazol-2-yl]-2H-pyrazol-3-ylamine(474-86F): the title compound (30 mg) was prepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(128 mg, 0.39 mmol), 4-methylpiperazine (39 mg, 0.43 mmol) andtriethylamine (0.1 mL). MS (m/z, ES+): 393.1 (M+1, 100%). Yield=20%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(2-methoxyethyl)-amide (551-8): the title compound (33 mg) was preparedfrom crude 2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonylchloride (75 mg, 0.23 mmol), 2-methoxyethylamine (39 mg, 0.43 mmol) andthe polystyrene resin bound base N-3-(morpholino)propyl polystyrenesulfonamide (PS-NMM) (0.24 g, 0.45 mmol) in 4 mL of methanol. MS (m/z,ES+): 368.5 (M+1, 100%); ¹H NMR (500 MHz, ppm, DMSO-d₆): δ 11.8-12.1 (brs, 1H), 8.5 (s, 1H), 7.99 (br d, 1H), 7.83 (d, 1H), 7.73 (t, 1H),5.9-6.7 (br s, 2H), 3.30 (t, 2H), 3.15 (s, 3H), 2.94 (dt, 2H), 2.41 (brs, 3H). Yield=46%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid4-fluoro-benzylamide (551-9): the title compound (8 mg) was preparedfrom crude 2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonylchloride (75 mg, 0.23 mmol), 4-fluorobenzylamine (29 μL, 0.25 mmol) andPS-NMM (0.22 g, 0.45 mmol) in 4 mL of chloroform. MS (m/z, ES+): 418.3(M+1, 100%). Yield=9%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(2-thiophen-2-ylethyl)-amide (551-10): the title compound (8 mg) wasprepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(75 mg, 0.23 mmol), 2-thiophen-2-ylethylamine (29 μL, 0.25 mmol) andPS-NMM (0.22 g, 0.45 mmol) in 4 mL of chloroform. MS (m/z, ES+): 420.4(M+1, 100): ¹H NMR (500 MHz, ppm, DMSO-d₆): δ 11.8-12.2 (br s, 1H), 8.5(s, 1H), 8.0 (d, 1H), 7.8 (m, 2H), 7.32 (d, 1H), 6.93 (dd, 1H), 6.86 (d,1H), 6.0 (br s, 2H), 3.03 (q, 2H), 2.92 (t, 2H), 2.43 (br s, 3H).Yield=8%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid4-chlorobenzylamide (551-11B): the title compound (21 mg) was preparedfrom crude 2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonylchloride (100 mg, 0.30 mmol), 4-chlorobenzylamine (74 μL, 0.60 mmol) andPS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 434.4(Cl³⁵ M+1, 100%), 436.4 (Cl³⁷ M+1, 40%). Yield=16%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid4-methoxybenzylamide (551-12B): the title compound (15 mg) was preparedfrom crude 2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonylchloride (100 mg, 0.30 mmol), 4-methoxybenzylamine (79 μL, 0.60 mmol)and PS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+):430.5 (M+1, 100%). Yield=11%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acidbenzylamide (551-13B): the title compound (16 mg) was prepared fromcrude 2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonylchloride (100 mg, 0.30 mmol), benzylamine (66 μL, 0.60 mmol) and PS-NMM(0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 400.5 (M+1,100%). Yield=13%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acidphenethylamide (551-14B): the title compound (9 mg) was prepared fromcrude 2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonylchloride (100 mg, 0.30 mmol), phenethylamine (76 μL, 0.60 mmol) andPS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 414.5(M+1, 100%). Yield=7%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid[2-(4-aminophenyl)-ethyl]-amide (551-15B): the title compound (25 mg)was prepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), phenethylamine (80 μL, 0.60 mmol) and PS-NMM (0.320g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 429.5 (M+1, 100%).Yield=19%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(2-morpholin-4-ylethyl)-amide (551-16): the title compound (56 mg) wasprepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), 2-morpholin-4-ylethylamine (80 μL, 0.60 mmol) andPS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 423.5(M+1, 100%). Yield=43%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(2,2,2-trifluoroethyl)-amide (551-17B): the title compound (11 mg) wasprepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), 2,2,2-trifluoroethylamine (48 μL, 0.60 mmol) andPS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 392.4(M+1, 100%). Yield=9%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acidcyclopropylmethylamide (551-18B): the title compound (27 mg) wasprepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), C-cyclopropylmethylamine (53 μL, 0.60 mmol) andPS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 364.5(M+1, 100%). Yield=21%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid[2-(3H-imidazol-4-yl)-ethyl]-amide (551-19B): the title compound (8 mg)was prepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), 2-(3H-imidazol-4-yl)-ethylamine (68 mg, 0.60 mmol)and PS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+):404.3 (M+1, 100%). Yield=6%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid4-aminobenzylamide (551-20): the title compound (15 mg) was preparedfrom crude 2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonylchloride (100 mg, 0.30 mmol), 4-aminomethylphenylamine (69 μL, 0.60mmol) and PS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z,ES+): 415.4 (M+1, 100%). Yield=12%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(pyridin-4-ylmethyl)-amide (551-21): the title compound (13 mg) wasprepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), C-pyridin-4-ylmethylamine (62 μL, 0.60 mmol) andPS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 401.3(M+1, 100%). Yield=11%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(3-dimethylamino-propyl)-amide (551-22C): the title compound (2 mg) wasprepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), N¹,N¹-dimethylpropane-1,3-diamine (77 μL, 0.60mmol) and PS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z,ES+): 395.3 (M+1, 100%). Yield=2%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(acetic hydrazido) amide (551-23A): the title compound (13 mg) wasprepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), acetic acid hydrazide (45 mg, 0.60 mmol) and PS-NMM(0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 367.3 (M+1,100%). Yield=12%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(2-dimethylamino-ethyl)-amide (551-24D): the title compound (11 mg) wasprepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), N¹,N¹-dimethylethane-1,2-diamine (67 μL, 0.60 mmol)and PS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+):381.4 (M+1, 50%). Yield=9%.

N-{2-[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonylamino]-ethyl}-acetamide(551-25B): the title compound (52 mg) was prepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), N-(2-aminoethyl)-acetamide (58 μL, 0.60 mmol) andPS-NMM (0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 395.6(M+1, 100%). Yield=43%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazole-6-sulfonic acid(phenylhydrazino) amide (551-26B): the title compound (32 mg) wasprepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride(100 mg, 0.30 mmol), phenylhydrazine (66 μL, 0.60 mmol) and PS-NMM(0.320 g, 0.60 mmol) in 5 mL of methanol. MS (m/z, ES+): 401.4 (M+1,100%). Yield=26%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-6-sulfonicacid amide (574-14): The title compound (30 mg) was prepared in twosteps from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-6-sulfonicacid, which had been derived from the chlorosulfonation of 90 mg of4-(5-fluorobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine, and asolution of excess ammonia dissolved in ethanol. MS (m/z, ES+): 328.0(M+1, 100%). Yield=25%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-6-sulfonicacid (2-hydroxy-ethyl)-amide (574-22A) and2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-4-sulfonicacid (2-hydroxy-ethyl)-amide (574-22AA): The title compounds wereprepared in two steps from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-6-sulfonylchloride, which had been derived from the chlorosulfonation of 50 mg of4-(5-fluorobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine, 183 mg(0.30 mmol) of ethanolamine and triethylamine (0.50 mL) in chloroform.The crude material was purified by flash column chromatography elutingwith CHCl₃:MeOH=9:1 to yield 12 mg (21%) of2-(5-amino-3-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-6-sulfonicacid (2-hydroxy-ethyl)-amide. MS (m/z, ES+): 372.0 (M+1, 100%).2-(5-amino-3-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-4-sulfonicacid (2-hydroxy-ethyl)-amide was isolated in a yield of 1 mg (2%). MS(m/z, ES+): 372.0 (M+1, 100%).

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-6-sulfonicacid (pyridin-4-ylmethyl)-amide (574-22B): The title compound (15 mg)was prepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-6-sulfonylchloride, which had been derived from the chlorosulfonation of 90 mg of4-(5-fluorobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine, 32 mg (0.30mmol) of C-pyridin-4-ylmethylamine, and triethylamine (0.50 mL) inchloroform. MS (m/z, ES+): 419.1 (M+1, 100%), 210.0 (100%). Yield=24%.

2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-4-fluorobenzothiazole-6-sulfonicacid amide (598-49): The title compound (280 mg) was prepared from crude2-(5-amino-3-methyl-1H-pyrazol-4-yl)-4-fluorobenzothiazole-7-sulfonylchloride, which had been derived from the chlorosulfonation of 320 mg of4-(4-fluorobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine, and asolution of excess ammonia dissolved in ethanol. MS (m/z, ES+): 328.0(M+1, 100%). Yield=72%.

Example 13 Synthesis of4-Benzothiazol-2-yl-5-phenyl-1H-pyrazol-3-ylamine

To a solution of 2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile(278 mg, 1 mmol) in anhydrous THF (40 mL) was addedphenylmagnesiumbromide (1 mmol) which was prepared from bromobenzene(152 mg, 1 mmol) and magnesium (25 mg, 1 mmol) in 10 mL of anhydrousTHF. The mixture was stirred at room temperature for 60 minutes and thenat 50° C. for 60 minutes. The resulting mixture was poured into asaturated ammonium chloride solution. The aqueous phase was extractedwith ethyl acetate. The extract was washed, dried over magnesiumsulfate, and evaporated. The residue was purified by flashchromatography eluting with hexanes:EtOAc=1:0-4:1 to yield 50 mg (16%)of the desired compound. MS (m/z, ES+): 309 (M+1, 100%).

The mixture of above prepared intermediate (50 mg, 0.16 mmol) andhydrazine hydrate (20 mg, 0.4 mmol) was refluxed in ethanol for 6 hours.The solvent was evaporated, and the resulting residue was purified byflash chromatography eluting with CH₂Cl₂:MeOH=1:0-9:1 to yield a yellowsolid. The solid was stirred in diethyl ether and an insoluble materialwas removed by filtration. The ether phase was concentrated to yield 25mg (53%) of the title compound as a yellow solid. MS (m/z, ES+): 293(M+1, 100%).

Example 14 Synthesis of4-Benzothiazol-2-yl-5-cyclopropyl-1H-pyrazol-3-ylamine

4-Benzothiazol-2-yl-5-cyclopropyl-1H-pyrazol-3-ylamine (23 mg) wasprepared using the procedure as described in Example 13 starting from556 mg (2.0 mmol) of2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile.Cyclopropylmagnesium bromide, which was prepared from cyclopropylbromide (484 mg, 4 mmol) and magnesium (100 mg, 4 mmol) in 8 mL ofanhydrous THF was added until the starting material had been consumed asdetermined by TLC. The title compound was isolated in a yield of 5%. MS(m/z, ES+): 257 (M+1, 100%).

Example 15 Synthesis of4-Benzothiazol-2-yl-5-pyridin-3-yl-1H-pyrazol-3-ylamine

1. A yellow suspension of 3-pyridinyl lithium in THF was preparedaccording to the literature (Cama, L. D.; Wildonger, K. J.; Guthikonda,R.; Ratcliffe, R. W.; Christensen, B. G. Tetrahedron (1983), 39, 2531)by adding n-BuLi (0.25 mL of a 2 M solution in cyclohexane, 0.5 mmol) toa solution of 3-bromopyridine (87 mg, 0.5 mmol) in anhydrous ether (3mL) at −78° C. To this slurry was rapidly added2-benzothiazol-2-yl-3,3-bismethylsulfanylacrylonitrile (140 mg, 0.5mmol) in anhydrous THF (5 mL) under argon. The resulting mixture wasstirred at −78° C. for 1 hour, and then slowly warmed up to roomtemperature and stirred at that temperature for 2 hours. Upon completionof the reaction, the mixture was poured into a saturated NH₄Cl aqueoussolution. The aqueous phase was extracted with ethyl acetate. Theorganic phase was washed with brine, dried over magnesium sulfate,filtered and evaporated. The crude material was then purified by flashchromatography eluting with CH₂Cl₂:MeOH=1:0-19:1 to yield 100 mg (65%)of the desired product as a brown oil. MS (m/z, ES+): 310 (M+1, 25%),263 (M−46, 100%).

2. The mixture of the above intermediate (100 mg, 0.32 mmol) andhydrazine hydrate (25 mg, 0.5 mmol) was heated at 70° C. in EtOH (10 mL)for 4 hours. The mixture was poured into a saturated NH₄Cl aqueoussolution. The aqueous phase was extracted with ethyl acetate. Theresulting brown oil was purified by preparative TLC twice eluting withCH₂Cl₂:MeOH=1:10 to yield 21 mg (22%) of the title compound as a creamcoloured solid. MS (m/z, ES+): 294 (M+1, 100%).

The following compounds were prepared in a manner analogous to theprocedure described in Example 15.

4-Benzothiazol-2-yl-5-pyridin-4-yl-2H-pyrazol-3-ylamine (549-68B): Thetitle compound (90 mg) was prepared in two steps starting from 560 mg(2.0 mmol) of 2-benzothiazol-2-yl-3,3-bis-methylsulfanyl-acrylonitrileand 390 mg (2.0 mmol) 4-bromopyridine hydrochloride. MS (m/z, ES+):294.08 (M+1, 100%). Yield=15%.

Example 16 Synthesis of4-Benzothiazol-2-yl-5-(4-nitrophenyl)-2H-pyrazol-3-ylamine

1. To a solution of benzothazole-2-acetonitrile (1.74 g, 10 mmol) inanhydrous dichloromethane (50 mL) was added triethylamine (1.95 g, 11mmol) and 4-nitrobenzoyl chloride (1.88 g, 10 mmol) in anhydrousdichloromethane (20 mL) at room temperature. The resulting mixture wasstirred at room temperature for 3 hrs. To the resulting suspension wasadded methanol (0.5 mL) and glacial acetic acid (0.5 mL). The resultingyellow solid was isolated by filtration and dried to yield 1.93 g (60%)of 2-benzothiazol-2-yl-3-hydroxy-3-(4-nitrophenyl)-acrylonitrile. MS(m/z, ES+): 324.0 (M+1, 100%).

2. The above prepared2-benzothiazol-2-yl-3-hydroxy-3-(4-nitrophenyl)-acrylonitrile (500 mg,1.55 mmol) was suspended in POCl₃ (12 mL). The suspension was heated at100° C. for 4 hrs and was then poured into ice water (25 mL). The pH ofthe aqueous phase was adjusted to neutral. The solids were isolated byfiltration and dried to yield 505 mg (95%) of2-benzothiazol-2-yl-3-chloro-3-(4-nitrophenyl)-acrylonitrile. MS (m/z,ES+): 341.92 (Cl³⁵M+1, 100%), 343.92 (Cl³⁷M+1, 45%).

3. The 2-benzothiazol-2-yl-3-chloro-3-(4-nitrophenyl)-acrylonitrile (500mg, 1.46 mmol) prepared above and hydrazine hydrate (150 mg, 3 mmol)were refluxed in ethanol (15 mL) for 6 hrs. The solvent was evaporatedand the residue was purified by flash column chromatography eluting withCH₂Cl₂:MeOH=9:1 to yield 182 mg (37%) of the title compound as a yellowsolid. MS (m/z, ES+): 338.04 (M+1, 100%).

The following compounds were prepared in a manner analogous to theprocedure described in Example 16.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-pyridin-4-yl-2H-pyrazol-3-ylamine(597-34): The title compound (260 mg) was prepared in three stepsstarting from 700 mg (3.15 mmol) of(5-fluoro-6-methoxy-benzothiazol-2-yl)-acetonitrile, 1.10 g (6.20 mmol)of isonicotinylchloride hydrochloride, 1.7 mL (12.2 mmol) oftriethylamine and a catalytic amount of DMAP. MS (m/z, ES+): 342.1 (M+1,100%). Yield=20%.

2-(5-Amino-3-pyridin-4-yl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acidamide (590-62): The title compound (5 mg) was prepared in three stepsstarting from 102 mg (0.40 mmol) of2-cyanomethylbenzothiazole-6-sulfonic acid amide, 70 mg (0.40 mmol) ofisonicotinylchloride hydrochloride and 228 mL (1.6 mmol) oftriethylamine. MS (m/z, ES+): 373.0 (M+1, 100%). Yield=3%.

Example 17 Synthesis of5-(4-Aminophenyl)-4-benzothiazol-2-yl-2H-pyrazol-3-ylamine (549-82)

An aqueous solution of Raney Ni (1 mL) was added to a suspension of4-benzothiazol-2-yl-5-(4-nitrophenyl)-2H-pyrazol-3-ylamine (115 mg, 0.34mmol) in ethanol (20 mL). To the vigorously stirring solution was addedhydrazine hydrate dropwise in 3 portions (3×50 mg, 3 mmol in total), andthe resulting mixture was then stirred for 2 hrs. The solids wereremoved by filtration and the mother liquor was evaporated to afford thecrude material. The residue was purified by flash column chromatographyeluting with CH₂Cl₂:MeOH=9:1 to yield 26 mg (25%) of the title compoundas a cream coloured solid. MS (m/z, ES+): 308.1 (M+1, 100%).

Example 18 Synthesis ofN-[4-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-phenyl]-hydroxylamine(590-6)

To a solution of4-benzothiazol-2-yl-5-(4-nitrophenyl)-2H-pyrazol-3-ylamine (218 mg, 0.65mmol) in DMF (4 mL) and 95% ethanol (4 mL) at room temperature was addedunder rapid stirring a suspension of zinc powder (200 mg) in aqueousammonium chloride (0.15 g in 2 mL of water). The resulting mixture wasthen stirred for 2 hrs. The residual zinc was removed by filtration andthe mother liquor was poured into water (100 mL). A yellow solid wasremoved by filtration. The aqueous phase was extract with ethyl acetate(2×75 mL). The combined extracts were washed with water, brine, driedover anhydrous sodium sulfate, filtered and evaporated. The resultingcrude material was purified by preparative TLC eluting with 10:1CH₂Cl₂:MeOH=10:1 to yield 7 mg (4%) of the title compound as creamcoloured solid. MS (m/z, ES+): 324.1 (M+1, 100%).

Example 19 Synthesis of4-Benzothiazol-2-yl-5-furan-2-yl-2H-pyrazol-3-ylamine (549-92)

1. To a solution of benzothazole-2-acetonitrile (700 mg, 4 mmol) inanhydrous dichloromethane (25 mL) was added triethylamine (1.2 g, 11.9mmol) and 2-furoyl chloride (522 mg, 4 mmol) dropwise at roomtemperature. The resulting mixture was stirred at room temperature for 5hrs. Dichloromethane (100 mL) was then added. The organic phase waswashed with 1% HCl aqueous solution, water, brine, and dried overanhydrous magnesium sulfate, filtered and evaporated. The resultingcrude material was washed with methanol and dried to afford 590 mg (55%)of furan-2-carboxylic acid2-benzothiazol-2-yl-2-cyano-1-furan-2-yl-vinyl ester as a yellow solid.MS (m/z, ES+): 363.01 (M+1, 100%).

2. A mixture of furan-2-carboxylic acid2-benzothiazol-2-yl-2-cyano-1-furan-2-yl-vinyl ester (660 mg, 1.82 mmol)and KOH (108 mg, 1.93 mmol) in ethanol (20 mL) was stirred first at roomtemperature overnight and then at 50° C. for 2 hours. The resultingsuspension was poured into water (200 mL), and the solution was adjustedto pH 7 using 5% HCl solution. The solids were isolated by filtration,washed with water, and air-dried to yield 474 mg (97%) of crude2-benzothiazol-2-yl-3-furan-2-yl-3-hydroxy acrylonitrile which was usedin the next step without further purification. MS (m/z, ES+): 268.98(M+1, 100%).

3. 2-Benzothiazol-2-yl-3-furan-2-yl-3-hydroxyacrylonitrile (474 mg, 1.77mmol) was suspended in POCl₃ (5 mL). The suspension was first stirred at50° C. for 1 hr then refluxed for 30 min until the solid dissolvedcompletely. The resulting brown solution was poured over crushed ice(200 mL). The resulting solids were isolated by filtration, washed withwater until the pH of the wash water became neutral, and air-dried toyield 477 mg (94%) of2-benzothiazol-2-yl-3-chloro-3-furan-2-ylacrylonitrile. MS (m/z, ES+):287.0 (Cl³⁵M+1, 100%), 289.0 (Cl³⁷M+1, 45%).

4. The 2-benzothiazol-2-yl-3-chloro-3-furan-2-ylacrylonitrile (480 mg,1.7 mmol) prepared above and hydrazine hydrate (100 mg, 2 mmol) wererefluxed in methanol (15 mL) for 3 hrs. The solvent was then evaporatedand the residue was purified by flash column chromatography eluting withCH₂Cl₂:MeOH=9:1 to afford 130 mg of material. This was further purifiedby preparative TLC eluting with CH₂Cl₂:MeOH=9:1 to yield 23 mg (5%) ofthe title compound. MS (m/z, ES+): 283.0 (M+1, 100%).

The following compounds were prepared in a manner analogous to theprocedure described in Example 19.

4-Benzothiazol-2-yl-5-thiophen-2-yl-2H-pyrazol-3-ylamine (590-42): Thetitle compound (30 mg) was prepared in four steps starting from 870 mg(5 mmol) of benzothazole-2-acetonitrile and 1.6 g (11 mmol) of2-thiophenecarbonyl chloride. MS (m/z, ES+): 299.0 (M+1, 100%).Yield=19%.

Example 20 Synthesis of4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-furan-2-yl-2H-pyrazol-3-ylamine(549-60)

1. A solution of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile (150mg, 0.675 mmol), 2-furoyl chloride (135 μL, 1.37 mmol) and triethylamine(235 μL, 1.69 mmol) in anhydrous dichloromethane was stirred at roomtemperature for approximately three hours forming a dark brown solution.The crude product, furan-2-carboxylic acid2-cyano-2-(5-fluoro-6-methoxybenzothiazol-2-yl)-1-furan-2-yl-vinylester, was identified by mass spectrometry. MS (m/z, ES+): 410.97 (M+1,80%), 520.98 (100%).

2. To the above reaction mixture was added a catalytic amount of DMAP(˜10 mg). The mixture was stirred for 2 hrs at room temperature. Afterthe reaction was complete, as indicated by TLC analysis, a small amountof concentrated HCl (˜1 mL) was added to precipitate the enol. Theproduct was then isolated by filtration and washed with 10% methanol indichloromethane to yield 175 mg (73%) of2-(5-fluoro-6-methoxybenzothiazol-2-yl)-3-furan-2-yl-3-hydroxyacrylonitrileas an off white solid. MS (m/z, ES+): 317 (M+1, 100%).

3. To a suspension of2-(5-fluoro-6-methoxybenzothiazol-2-yl)-3-furan-2-yl-3-hydroxyacrylonitrile(70 mg, 0.199 mmol) in anhydrous dichloromethane (3 mL) and carbontetrachloride (2 mL) was added triphenylphosphine (175 mg, 0.667 mmol).The reaction mixture was stirred at reflux for three hours then at roomtemperature overnight to give a dark brown solution containing3-chloro-2-(5-fluoro-6-methoxybenzothiazol-2-yl)-3-furan-2-ylacrylonitrile.Hydrazine monohydrate (30 μL, 0.62 mmol) and methanol (1 mL) were thenadded and the solution was refluxed for 2 hrs. At this point, conc. HCl(0.7 mL) was added and the solution was refluxed for an additional hour.The mixture was then cooled to room temperature and neutralized withammonium hydroxide. The solvent was evaporated and the crude product waspurified by flash column chromatography eluting with CH₂Cl₂:MeOH=9:1 toyield 15 mg (21%) of the title compound as a brown solid. MS (m/z, ES+):331.1 (M+1, 100%).

Example 21 Synthesis of5-Cyclopropyl-4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-2H-pyrazol-3-ylamine(597-65)

To a 10 mL reaction vessel containing(5-fluoro-6-methoxybenzothiazol-2-yl) acetonitrile (100 mg, 0.45 mmol)in 4 mL of anhydrous dichloromethane were added cyclopropane carbonylchloride (21 μL, 0.231 mmol), 1.35 equivalents of triethylamine (85 μL)and a catalytic amount of DMAP (10 mg). The reaction was agitated atroom temperature for approximately 3 hrs. At this point the solvent wasevaporated and CCl₄ (4 mL), 2.5 equivalents of polystyrene resin boundtriphenylphosphine (725 mg, 1.55 mmol/g), and triethylamine (85 μL) wereadded. After agitation at 65° C. for a minimum of 3 hrs, the reactionmixture was concentrated. Ethanol (4 mL) and hydrazine monohydrate (40μL, 1.8 equivalents) were then added and the mixture was refluxed forapproximately 6 hrs. Several drops of concentrated HCl were then addedto ensure complete cyclization of the pyrazole ring. After completion ofthe reaction, the solids were removed by filtration and the resin waswashed three times with 10% methanol in dichloromethane. The filtratewas then concentrated and purified by flash column chromatographyeluting with CH₂Cl₂:MeOH=50:1 to yield 40 mg (29%) of the title compoundas a cream coloured solid. MS (m/z, ES+): 305.1 (M+1, 100%)

The following compounds were prepared in a manner analogous to theprocedure described in Example 21.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-phenyl-2H-pyrazol-3-ylamine(597-67A): The title compound (48 mg) was prepared starting from 100 mg(0.45 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 58μL (0.50 mmol) of benzoyl chloride. MS (m/z, ES+): 341.1 (M+1, 100%).Yield=31%.

5-(2-Chloropyridin-3-yl)-4-(5-fluoro-6-methoxybenzothiazol-2-yl)-2H-pyrazol-3-ylamine(597-67C): The title compound (26 mg) was prepared starting from 100 mg(0.45 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 87mg (0.50 mmol) of 2-chloronicotinoyl chloride. MS (m/z, ES+): 376.1(Cl³⁵M+1, 100%), 378.1 (Cl³⁷M+1, 40%). Yield=15%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-(4-fluorophenyl)-2H-pyrazol-3-ylamine(597-67D): The title compound (26 mg) was prepared starting from 100 mg(0.45 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 59μL (0.50 mmol) of 4-fluorobenzoyl chloride. MS (m/z, ES+): 359.1 (M+1,100%). Yield=28%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-(3-fluorophenyl)-2H-pyrazol-3-ylamine(597-67E): The title compound (37 mg) was prepared starting from 100 mg(0.45 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 59μL (0.50 mmol) of 3-fluorobenzoyl chloride. MS (m/z, ES+): 359.1 (M+1,100%). Yield=22%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine(597-69A): The title compound (32 mg) was prepared starting from 80 mg(0.36 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 68mg (0.4 mmol) of 4-methoxybenzoyl chloride. MS (m/z, ES+): 371.1 (M+1,100%). Yield=24%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-isoxazol-5-yl-2H-pyrazol-3-ylamine(597-69F): The title compound (5 mg) was prepared starting from 80 mg(0.36 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 38μL (0.4 mmol) of 5-isoxazolecarbonyl chloride. MS (m/z, ES+): 332.0(M+1, 100%), 318.0 (enol, 30%). Yield=11%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-(3-nitrophenyl)-2H-pyrazol-3-ylamine(597-69B): The title compound (24 mg) was prepared starting from 80 mg(0.36 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 74mg (0.4 mmol) of 3-nitrobenzoyl chloride. MS (m/z, ES+): 386.1 (M+1,100%). Yield=17%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-(4-fluorophenyl)-2H-pyrazol-3-ylamine(597-69D): The title compound (26 mg) was prepared starting from 80 mg(0.36 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 47μL (0.4 mmol) of 4-fluorobenzoyl chloride. MS (m/z, ES+): 359.0 (M+1,100%). Yield=20%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-thiophen-2-yl-2H-pyrazol-3-ylamine(597-69E): The title compound (32 mg) was prepared starting from 80 mg(0.36 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 42μL (0.4 mmol) of 2-thiophenecarbonyl chloride. MS (m/z, ES+): 347.0(M+1, 100%). Yield=25%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-(5-mitrofuran-2-yl)-2H-pyrazol-3-ylamine(597-69G): The title compound (12 mg) was prepared starting from 80 mg(0.36 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 70mg (0.4 mmol) of 5-nitro-2-furoyl chloride. MS (m/z, ES+): 376.0 (M+1,100%). Yield=9%.

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-(2-phenylcyclopropyl)-2H-pyrazol-3-ylamine(597-71B): The title compound (5 mg) was prepared starting from 100 mg(0.45 mmol) of (5-fluoro-6-methoxybenzothiazol-2-yl)-acetonitrile and 75μL (0.48 mmol) of trans-2-phenylcyclopropylcarbonyl chloride. MS (m/z,ES+): 381.1 (M+1, 100%). Yield=4%.

Example 22 Synthesis of3-(5-Amino-4-benzothiazol-2-yl-2H-pyrazol-3-yl)-propan-1-ol

1. To a solution of benzothazole-2-acetonitrile (870 mg, 5 mmol) inanhydrous dichloromethane (50 mL) were first added triethylamine (1.11g, 11 mmol), and then 4-bromobutyryl chloride (1.85 g, 10 mmol) dropwiseat room temperature. The resulting mixture was stirred at roomtemperature for 4 hrs. Additional triethylamine (0.22 g) and4-bromobutyryl chloride (0.48 g) were added and stirring was continuedfor an additional 2 hrs. The reaction solution was diluted withdichloromethane (100 mL). The organic phase was washed with 0.1% NaOH,water, brine, dried over anhydrous sodium sulfate, filtered andevaporated. The resulting crude material was washed with methanol anddried to afford 97 mg (6%) ofbenzothiazol-2-yl-(dihydrofuran-2-ylidene)-acetonitrile. MS (m/z, ES+):343 (M+1, 100%).

2. The benzothiazol-2-yl-(dihydrofuran-2-ylidene)-acetonitrile (90 mg,0.37 mmol) prepared above and hydrazine hydrate (25 mg, 0.5 mmol) wererefluxed in ethanol (15 mL) for 10 hours. The solvent was evaporated andthe resulting residue was triturated with dichloromethane. The solid wasisolated by filtration and was then washed with dichloromethane andmethanol to yield 60 mg (67%) of the title compound as a cream colouredsolid. MS (m/z, ES+): 275.1 (M+1, 100). ¹H NMR (300 MHz, ppm, DMSO-d₆):δ (ppm), 11.5-12.2 (br s, 1H), 8.00 (d, 1H), 7.86 (d, 1H), 7.77 (t, 2H),7.29 (t, 1H), 7.00-5.20 (br m, 2H), 4.55 (s, 1H), 3.52 (q, 2H), 2.83 (brs, 2H), 1.84 (q, 2H).

The following compounds were prepared in a manner analogous to theprocedure described in Example 22.

4-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-butan-1-ol: The titlecompound (47 mg) was prepared starting from 522 mg (3 mmol) ofbenzothazole-2-acetonitrile and 538 mg (3.6 mmol) of 5-bromopentanoylchloride. MS (m/z, ES+): 289.1 (M+1, 100%). Yield=48%.

Example 23 Synthesis of4-Benzothiazol-2-yl-5-(3-methylamino-propyl)-2H-pyrazol-3-ylamine

1. To a solution of concentrated H₂SO₄ (3 mL) and hydrobromic acid (6 mLof 48%) was added3-(5-amino-4-benzothiazol-2-yl-2H-pyrazol-3-yl)-propan-1-ol (810 mg, 3.0mmol). The resulting mixture was refluxed for 2 hrs. The mixture wascooled to room temperature, poured into ice water, and adjusted to pH3-4 using 5% NaOH solution. The resulting sticky solid was isolated byfiltration, washed with water and air-dried to yield 815 mg (80%) of4-benzothiazol-2-yl-5-(3-bromopropyl)-2H-pyrazol-3-ylamine as a creamcoloured solid. The crude material was used in the subsequent stepwithout further purification. MS (m/z, ES+): 337.1 (Br⁷⁹M+1, 61%), 339.1(Br⁸¹M+1, 59%), 257.2 (M-HBr, 100%).

2. A mixture of4-benzothiazol-2-yl-5-(3-bromopropyl)-2H-pyrazol-3-ylamine (130 mg, 0.39mmol) and methylamine (5 mL of a 2 M solution in THF, 10 mmol) wasstirred at room temperature for 60 hrs. The resulting solid was isolatedby filtration, washed with THF, and then with water. The product was airdried to yield 38 mg (32%) of the title compound as an off white solid.MS (m/z, ES+): 288.2 (M+1, 100%).

The following compounds were prepared in a manner analogous to theprocedure described in Example 23.

5-(3-Aminopropyl)-4-benzothiazol-2-yl-2H-pyrazol-3-ylamine (590-86A):The title compound (12 mg) was prepared using 130 mg (0.39 mmol) of4-benzothiazol-2-yl-5-(3-bromopropyl)-2H-pyrazol-3-ylamine, which wasderived from3-(5-amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-propan-1-ol, and asaturated solution of ammonia gas in ethanol. MS (m/z, ES+): 274.2 (M+1,100%), 257.2 (M-NH2, 70%).

4-Benzothiazol-2-yl-5-(3-dimethylaminopropyl)-2H-pyrazol-3-ylamine(590-87A): The title compound (100 mg) was prepared using 200 mg (0.59mmol) of 4-benzothiazol-2-yl-5-(3-bromopropyl)-2H-pyrazol-3-ylamine,which was derived from3-(5-amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-propan-1-ol, and 5 mL ofa 2 M solution of dimethylamine in THF (10 mmol). MS (m/z, ES+): 302.1(M+1, 100%).

4-Benzothiazol-2-yl-5-(4-methylaminobutyl)-2H-pyrazol-3-ylamine(590-94): The title compound (30 mg) was prepared using 185 mg (0.39mmol) of 4-benzothiazol-2-yl-5-(4-bromobutyl)-2H-pyrazol-3-ylamine,which was derived from4-(5-amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)butan-1-ol, and 5 mL of a2 M solution of methylamine in THF (10 mmol). MS (m/z, ES+): 302.4 (M+1,100%).

4-Benzothiazol-2-yl-5-(4-dimethylaminobutyl)-2H-pyrazol-3-ylamine(590-95): The title compound (41 mg) was prepared using 185 mg (0.39mmol) of 4-benzothiazol-2-yl-5-(4-bromobutyl)-2H-pyrazol-3-ylamine,which was derived from4-(5-amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-butan-1-ol, and 5 mL ofa 2 M solution of dimethylamine in THF (10 mmol). MS (m/z, ES+): 316.4(M+1, 100%).

Example 24 Synthesis of4-Benzothiazol-2-yl-5-piperidin-4-yl-2H-pyrazol-3-ylamine (610-48)

1. To a solution of benzothazole-2-acetonitrile (700 mg, 4 mmol),triethylamine (1.1 g, 10.9 mmol), and a catalytic amount of DMAP, in 60mL of anhydrous dichloromethane at room temperature under argon, wasadded 1-acetylpiperidine-4-carbonylchloride hydrochloride (1.1 g, 4.46mmol) in small portions over 2 hrs. The resulting mixture was stirred atroom temperature overnight. The reaction mixture was diluted withdichloromethane. The organic phase was washed with water, brine, driedover anhydrous sodium sulfate, filtered, and evaporated. The resultingcrude product was purified by flash column chromatography eluting withCH₂Cl₂:MeOH=20:1 to yield 880 mg (67%) of3-(1-acetylpiperidin-4-yl)-2-benzothiazol-2-yl-3-hydroxyacrylonitrile asa light brown solid. MS (m/z, ES+): 328.1 (M+1, 100%).

2. To a solution of the above prepared3-(1-acetylpiperidin-4-yl)-2-benzothiazol-2-yl-3-hydroxyacrylonitrile(415 mg, 1.27 mmol) in anhydrous dichloromethane (20 mL) at roomtemperature under argon were added triethylamine (193 mg, 1.9 mmol) andtosyl chloride (303 mg, 1.59 mmol) in small portions. The resultingmixture was stirred at room temperature for 6 hrs. The reaction mixturewas diluted with dichloromethane. The organic phase was washed with 1%HCl, 0.5% NaOH, water, brine, and dried over anhydrous sodium sulfate,filtered, and evaporated. The resulting crude product was purified byflash column chromatography eluting with CH₂Cl₂:MeOH=20:1 to yield 140mg (23%) of toluene-4-sulfonic acid1-(1-acetylpiperidin-4-yl)-2-benzothiazol-2-yl-2-cyanovinyl ester. MS(m/z, ES+): 482.1 (M+1, 100%).

3. A mixture of the above prepared toluene-4-sulfonic acid1-(1-acetylpiperidin-4-yl)-2-benzothiazol-2-yl-2-cyanovinyl ester (140mg, 0.29 mmol) and hydrazine hydrate (25 mg, 0.5 mmol) in methanol wasrefluxed overnight. The solvent was evaporated and the residue waspurified by flash column chromatography eluting with CH₂Cl₂:MeOH=20:1 toyield 54 mg (55%) of1-[4-(5-amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-piperidin-1-yl]-ethanone.MS (ES+): 342.1 (M+1, 100%).

4. A solution of the above prepared1-[4-(5-amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-piperidin-1-yl]-ethanone(54 mg, 0.158 mmol) in 6N HCl (10 mL) was refluxed for 6 hrs and wasallowed to cool to room temperature overnight. The resulting mixture waspoured over crushed ice (20 mL) and the solution was adjusted to neutralpH using a 10% NaOH solution. The resulting solution was saturated withNaCl and was extracted with ethyl acetate (10×50 mL). The combinedextracts were washed once with brine, dried over anhydrous sodiumsulfate, filtered and evaporated. The resulting crude product waspurified by flash column chromatography eluting with CH₂Cl₂:MeOH=20:1with 1% ammonia to yield 15 mg (32%) of the title compound. MS (m/z,ES+): 300.14 (M+1, 100%).

Example 25 2-(3-Phenyl-1H-pyrazol-4-yl)benzothiazole

To a solution of 2-methylbenzothiazole (149 mg, 1 mmol) in THF was addedn-BuLi (0.5 mL of a 2M solution in cyclohexane, 1 mmol) at −70° C. Themixture was stirred at this temperature for 1.5 hours and then ethylbenzoate (150 mg, 1 mmol) was added dropwise. After stirring for another1.5 hours, a saturated solution of NH₄Cl was added dropwise. Thereaction was then allowed to warm to 5° C. and a precipitate obtainedwas isolated by filtration and washed with water. The resulting crudematerial was recrystallized from methanol to yield the desiredintermediate.

A solution of the above prepared intermediate (147 mg, 0.58 mmol) andDMF dimethyl acetal (76 mg, 0.64 mmol) in 5 mL of toluene was stirredovernight at room temperature and was then heated to reflux for 2 hours.The mixture was concentrated in vacuo and the residue was purified byrecrystallization to afford the desired product.

The above-prepared intermediate (86 mg, 0.28 mmol) was dissolved inmethanol (10 mL) and then treated with hydrazine hydrate (30 mg, 0.6mmol). The mixture was stirred at room temperature for 16 hours. At thispoint, the solvent was removed in vacuo and the residue was purified byrecrystallization to afford 71 mg of the title compound. MS (m/z, ES+):278 (M+1, 100%); ¹H NMR (400 MHz, ppm, CDCl₃): δ 13.57 (br s, 1H), 8.6and 8.1 (br, 1H), 7.99 (d, 1H), 7.91 (d, 1H), 7.72-7.66 (m, 2H),7.55-7.40 (m, 4H), 7.35 (dd, 1H). Yield=27%.

The following compounds were prepared in a manner analogous to theprocedure described in Example 25.

2-[3-(4-Methoxyphenyl)-1H-pyrazol-4-yl]benzothiazole: The title compound(457 mg) was prepared in three steps starting from 1.49 g (10 mmol) of2-methylbenzothiazole and 1.66 g (10 mmol) of methyl 4-methoxybenzoate.MS (m/z, ES+): 308 (M+1, 100%). ¹H NMR (400 MHz, ppm, CDCl₃): δ 8.28 (s,1H), 8.00 (d, ³J=8.1 Hz, 1H), 7.75 (d, ³J=8.0 Hz, 1H), 7.57 (d, ³J=8.6Hz, 2H), 7.44 (dd, ³J=7.4 Hz, ³J=8.1 Hz, 1H), 7.33 (dd, ³J=7.4 Hz,³J=8.0 Hz, 1H), 7.00 (d, ³J=8.6 Hz, 2H), 3.87 (s, 3H). Yield=18%.

2-[3-(2-Methoxyphenyl)-1H-pyrazol-4-yl]benzothiazole: The title compound(430 mg) was prepared in four steps starting from 1.49 g (10 mmol) of2-methylbenzothiazole and 1.66 g (10 mmol) of methyl 2-methoxybenzoate.After the reaction with hydrazine, the addition product was dissolved inmethanol and heated to reflux with a catalytic amount of p-TSA (15 mg)for 2 hours to yield the title compound. MS (m/z, ES+): 308 (M+1, 100%).Yield=14%.

2-(3-Methyl-1H-pyrazol-4-yl)-benzothiazole (515-84): The title compound(970 mg) was prepared in three steps starting from 2-methylbenzothiazoleand ethyl acetate. MS (m/z, ES+): 216.04 (M+1, 100%).

Example 26 Synthesis of 4-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)phenol

To a suspension of 2-[3-(4-methoxyphenyl)-1H-pyrazol-4-yl]benzothiazole(100 mg, 0.33 mmol) was slowly added tribromoborane (3.3 mL of a 1 Msolution in CH₂Cl₂, 3.3 mmol). The mixture was stirred overnight. Thereaction was then quenched by the addition of methanol. The mixture wasneutralized with sodium carbonate solution and extracted three timeswith ethyl acetate. The organic extracts were combined, washed withbrine, dried over Na₂SO₄, filtered, and evaporated. The crude materialwas recrystallized from ethyl acetate to yield 64 mg (66%) of the titlecompound as a yellow solid. MS (m/z, ES+): 294 (M+1, 100%). ¹H NMR (400MHz, ppm, DMSO-d₆): δ 13.5 (br s, 1H), 9.80 (br s, 1H), 8.22 (br s, 1H),7.98 (d, ³J=8.1 Hz, 1H), 7.91 (d, ³J=8.0 Hz, 1H), 7.47 (d, ³J=8.0 Hz,2H), 7.45 (dd, ³J=7.4 Hz, ³J=8.1 Hz, 1H), 7.34 (dd, ³J=7.4 Hz, ³J=8.0Hz, 1H), 6.89 (d, ³J=8.0 Hz, 2H).

Example 27 Synthesis of 2-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)phenol

2-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)phenol (82 mg) was prepared inanalogy to the procedure as described in Example 26 starting from 100 mg(0.33 mmol) of 2-[3-(2-methoxy-phenyl)-1H-pyrazol-4-yl]benzothiazole. MS(m/z, ES+): 294 (M+1, 100%). Yield=85%.

Example 28 Synthesis of4-Benzothiazol-2-yl-2-methyl-2H-pyrazol-3-ylamine

A solution of benzothiazol-2-ylacetonitrile (522 mg, 3 mmol) and DMFdimethyl acetal (394 mg, 3.3 mmol) in toluene (6 mL) was heated toreflux for 3 hours and then stirred overnight at room temperature. Themixture was concentrated and the residue was purified byrecrystallization from 2-propanol to yield 350 mg (51%) of the productas a pale yellow solid.

A mixture of the 2-benzothiazol-2-yl-3-dimethylaminoacrylonitrileprepared above (96 mg, 0.42 mmol) and triethylamine (0.3 mL) in EtOH (10mL) was treated with methylhydrazine sulfate (180 mg, 1.25 mmol) and themixture was heated to reflux for 2 days. The solution was concentratedin vacuo and the residue was purified by flash chromatography(hexanes:EtOAc=1:1) to yield 47 mg (49%) of the title compound as ayellow powder. MS (m/z, ES+): 231 (M+1, 100%).

Example 29 Synthesis of2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-benzothiazole-6-carboxylic acidmethyl ester (574-26E2)

A solution of2-(3-amino-5-methyl-1H-pyrazol-4-yl)-benzothiazole-6-carboxylic acid(1.52 g, 5.5 mmol) in 500 mL of anhydrous methanol containing HCl gaswas heated to reflux for 5 hrs. The excess methanol was removed bydistillation and the solution was neutralized by the addition of asolution of saturated sodium carbonate. The resulting solids wereisolated by filtration to yield 500 mg of the title compound as a yellowsolid. MS (m/z, ES+): 289.1 (M+1, 100%).

Example 30 Synthesis of2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-4,5,6-trifluorobenzothiazole-7-sulfonicacid amide (598-79)

5-Methyl-4-(4,5,6-trifluorobenzothiazol-2-yl)-2H-pyrazol-3-ylamine (474mg; 1.67 mmol) was heated at 140-150° C. in chlorosulfonic acid (4 ml)for 72 h. After cooling to room temperature, the mixture was poured overice, and the product was extracted into ethyl acetate. The combinedextracts were washed with water, dried over sodium sulfate, filtered andevaporated. The residue was dissolved in ethanol and a solution ofammonia in ethanol (15 ml) was added. The reaction mixture was stirredat room temperature for 1 hr, while ammonia gas was bubbled through themixture. The volume of solvent was then reduced evaporated and water wasadded. The resulting beige precipitate was filtered off and dried invacuum to yield 490 mg (81%) of the title compound. MS (m/z, ES+): 313.0(Cl³⁵M+1, 100%), 315.0 (Cl³⁷M+1, 50%); ¹H NMR (300 MHz, ppm, DMSO-d₆): δ11.75 (br s, 1H), 7.80 (d, 1H, J_(HF)=11.4 Hz), 6.25 (s, 2H), 3.95 (s,3H), 2.39 (s, 3H).

Example 31 Synthesis of4-(7-Chloro-5-fluoro-6-methoxybenzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine(598-80)

4-(5-Fluoro-6-methoxybenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine(596 mg; 2.14 mmol) was mixed with 4 mL of sulfuryl chloride. Thereaction was stirred at room temperature for several hours and then thereaction was quenched by the addition of water. The resultingprecipitate was isolated by filtration to yield 417 mg (62%) of thetitle compound. MS (m/z, ES+): 364.2 (M+1, 100%).

Example 32 Synthesis of2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazol-5-ol (574-13)

To a suspension of4-(5-methoxy-benzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine (60 mg,0.23 mmol) in CH₂Cl₂ (5 mL) at 4° C. was slowly added tribromoborane(2.3 mL of a 1M solution in dichloromethane, 2.3 mmol). The reactiontemperature was maintained at 4° C. and the solution was stirredovernight. The mixture was then neutralized with sodium carbonatesolution. The resulting solids were isolated by filtration and werepurified by flash chromatography eluting with CHCl₃:MeOH=9:1 to yield 10mg (19%) of the title compound as a cream coloured solid. MS (m/z, ES+):247.1 (M+1, 100%).

Example 33 Synthesis of[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)benzothiazol-5-yl]-methanol(574-21)

To a solution of2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-5-carboxylic acidethyl ester (30 mg) in THF (1 mL) was added lithium aluminum hydride (4mg). The reaction mixture was stirred at room temperature for 5 hrs atwhich point sodium sulfate nonahydrate was added. The resulting mixturewas stirred for an additional 30 min. The solids were removed byfiltration. The solvent was then evaporated and the residue was purifiedby flash column chromatography eluting with CHCl₃:MeOH=9:1 to yield 21mg (81%) of the title compound as a cream coloured solid. MS (m/z, ES+):261.1 (M+1, 100%).

The following compounds were prepared in a manner analogous to theprocedure described in Example 33.

[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-6-yl]-methanol(574-36A): The title compound (340 mg) was prepared starting from 470 mg(1.63 mmol) of2-(3-amino-5-methyl-1H-pyrazol-4-yl)-benzothiazole-6-carboxylic acidmethyl ester. The final product was purified by recrystallization froman ethanol/water mixture. MS (m/z, ES+): 261.0 (M+1, 100%). Yield=80%.

[2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazol-6-yl]-methanol(574-50A): The title compound (30 mg) was prepared starting from 54 mg(0.18 mmol) of2-(3-amino-5-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazole-6-carboxylicacid methyl ester. The final product was purified by recrystallizationfrom an ethanol/water mixture. MS (m/z, ES+): 279.1 (M+1, 100%).Yield=61%.

Example 34 Synthesis of5-Methyl-4-(6-pyrrolidin-1-ylmethyl-benzothiazol-2-yl)-2H-pyrazol-3-ylamine(574-36C)

1. To a solution of concentrated H₂SO₄ (0.3 mL) and hydrobromic acid(0.6 mL, 48%) was added[2-(5-amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-6-yl]-methanol (52mg, 0.2 mmol). The resulting mixture was refluxed for 2 hrs. Uponcooling to room temperature, the mixture was poured into ice waterresulting a milky solution. The suspension was neutralized to pH 3-4with 5% NaOH solution. The resulting cream coloured solid was isolatedby filtration, washed with water and air-dried to yield 58 mg of4-(6-bromomethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine. Thecrude material was used in the subsequent step without furtherpurification.

2. To a suspension of4-(6-bromomethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine (20 mg)in ethanol (1 mL) was added excess pyrrolidine. The reaction mixture wasstirred at room temperature overnight. The solvent was then removed byevaporation and the residue was purified by flash column chromatographyeluting with CHCl₃:MeOH=9:1 to yield 2.7 mg of the title compound as acream coloured solid. MS (m/z, ES+): 314.1 (M+1, 45%), 243.0 (M-C₄H₈N,100%).

The following compounds were prepared in a manner analogous to theprocedure described in Example 34.

5-Methyl-4-(6-methylaminomethylbenzothiazol-2-yl)-2H-pyrazol-3-ylamine(574-36E): The title compound (1.6 mg) was prepared starting from 18 mgof 4-(6-bromomethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine andexcess methylamine. MS (m/z, ES+): 274.1 (M+1, 100%), 243.1 (M-CH₃NH,60%).

5-Methyl-4-[6-(4-methylpiperazin-1-ylmethyl)-benzothiazol-2-yl]-2H-pyrazol-3-ylamine(574-36D): The title compound (9 mg) was prepared starting from 20 mg of4-(6-bromomethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine andexcess 1-methylpiperazine. MS (m/z, ES+): 343.1 (M+1, 20%), 244.1(M-CH₃N(CH₂)₄N+1, 100%).

5-Methyl-4-(6-morpholin-4-ylmethylbenzothiazol-2-yl)-2H-pyrazol-3-ylamine(574-38B): The title compound (27 mg) was prepared starting from 40 mgof 4-(6-bromomethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine andexcess morpholine. MS (m/z, ES+): 330.1 (M+1, 30%), 243 (M-O(CH₂)₄N,100%).

4-(6-Dimethylaminomethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine(574-38A): The title compound (24 mg) was prepared starting from 40 mgof 4-(6-bromomethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine andexcess dimethylamine. MS (m/z, ES+): 288.1 (M+1, 20%), 243.1 (M-(CH₃)₂N,100%).

2-{[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-6-ylmethyl]-amino}-ethanol(574-38C): The title compound (10 mg) was prepared starting from 40 mgof 4-(6-bromomethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine andexcess 2-aminoethanol. MS (m/z, ES+): 304.1 (M+1, 10%), 243.2(M-HOCH₂CH₂NH, 100%).

3-{[2-(5-Amino-1H-pyrazol-4-yl)-benzothiazol-6-ylmethyl]-amino}-N-methylbenzenesulfonamide(574-38F): The title compound (9 mg) was prepared starting from 40 mg of4-(6-bromomethylbenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine andexcess 3-amino-N-methylbenzenesulfonamide. MS (m/z, ES+): 429.2 (M+1,100%).

4-(6-Dimethylaminomethyl-5-fluorobenzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine(574-50C): The title compound (28 mg) was prepared in two steps startingfrom 30 mg (0.1 mmol) of[2-(3-amino-5-methyl-1H-pyrazol-4-yl)-5-fluorobenzothiazol-6-yl]methanol.The reaction of the bromide was carried out with excess dimethylamine.MS (m/z, ES+): 306.1 (M+1, 43%), 261.1 (60%), 153.5 (100%).

Example 354-Benzothiazol-2-yl-N⁵-(1H-imidazol-2-ylmethylene)-1H-pyrazole-3,5-diamine(610-49)

A mixture of 4-benzothiazol-2-yl-1H-pyrazole-3,5-diamine (50 mg, 0.22mmol) and 2-imidazolecarbonylaldehyde (22 mg, 0.22 mmol) in methanol (10mL) were refluxed overnight. The solvent was evaporated and theresulting crude material was purified by flash column chromatographyeluting with CH₂Cl₂:MeOH=20:1 to yield 21 mg (31%) of the titlecompound. MS (m/z, ES+): 310.1 (M+1, 15%), 232.1 (M−77, 60%), 142.0(M−167, 68%), 101.1 (100%).

Example 364-Benzothiazol-2-yl-N⁵-(1H-imidazol-2-ylmethyl)-1H-pyrazole-3,5-DIAMINE(610-52)

A mixture of4-benzothiazol-2-yl-N⁵-(1H-imidazol-2-ylmethylene)-1H-pyrazole-3,5-diamine(13 mg, 0.042 mmol) and NaBH₄ (10 mg) was stirred at room temperaturefor 3 hrs. The solvent was then evaporated and the residue was purifiedby flash column chromatography eluting with a gradient of CH₂Cl₂ toCH₂Cl₂:MeOH=4:1 to yield 12 mg (84%) of the title compound. MS (m/z,ES+): 312.1 (M+1, 80%), 232.1 (100%).

Example 37 Synthesis of 2-(1H-pyrazol-4-yl)-benzothiazole-6-sulfonicacid amide (523-88-39)

A solution of 2-(1H-pyrazol-4-yl)benzothiazole (600 mg, 0.30 mmol) inneat chlorosulfonic acid was heated to 150° C. for 5 hrs. The reactionmixture was then poured over ice and the resulting precipitate wasisolated by filtration to yield a mixture of2-(1H-pyrazol-4-yl)-benzothiazole-6-sulfonyl chloride and2-(1H-pyrazol-4-yl)-benzothiazole-4-sulfonyl chloride.

The crude material prepared above was reacted with ammonia hydroxide inethanol. The solvent was evaporated and the crude material was purifiedby flash column chromatography to yield 37 mg of the title compoundcontaminated with approximately 25% of2-(1H-pyrazol-4-yl)-benzothiazole-4-sulfonic acid amide as determined by¹H NMR. MS (m/z, ES+): 281.2 (M+1, 100%); ¹H NMR (300 MHz, ppm,DMSO-d₆): 13.50 (br s, 1H), 8.7 (d, 1H), 8.8 (br s, 2H), 8.08 (d, 1H),7.9 (dd, 1H), 7.42 (br s, 2H) (major isomer).

Example 38 Synthesis of(4-Benzothiazol-2-yl-2H-pyrazol-3-yl)-methylamine (523-32A)

A solution of 4-benzothiazol-2-yl-1H-pyrazol-3-ylamine (1.1 g, 5.0 mmol)in 20 mL of formic acid was heated to 70° C. overnight. The formic acidwas evaporated under reduced pressure and the resulting crude materialwas recrystallized from ethanol to yield 1.1 g (90%) ofN-(4-benzothiazol-2-yl-1H-pyrazol-3-yl)-formamide.

To the above prepared N-(4-benzothiazol-2-yl-1H-pyrazol-3-yl)-formamide(0.50 g, 2.0 mmol) in 50 mL of anhydrous THF at 0° C. was added 0.50 gof lithium aluminum hydride. The reaction was stirred for 1 hr and thenwas quenched by the addition of saturated ammonium chloride solution.The solids were removed by filtration. The solvent was then evaporatedand the crude product was purified by recrystallization from acetone toyield 280 mg (61%) of the title compound. MS (m/z, ES+): 231.1 (M+1,100%).

For the biological examples below, conditions are room temperatureunless otherwise stated.

Example 39 In Vitro Activity Profile for Kinases

Enzyme Preparation and Use

The target ILK is a full-length recombinant protein expressed in Hi5insect cells by baculovirus infection. Recombinant ILK protein wasexpressed using cultured insect cells and a baculovirus expressionsystem. Standard techniques for DNA manipulation were used to producerecombinant DNA molecules and baculoviruses (Sambrook. J., Fritsch, E.F. and Maniatis, T. 1989, Molecular cloning, a laboratory manual. Secondedition. Cold Spring Harbor Laboratory Press. NY; Crossen, R. andGruenwald, S. 1998. Baculovirus expression Vector System Manual. 5^(th)Edition. (Pharmingen, San Diego, Calif.). The ILK open reading frame(Hannigan et al., supra.), excluding the 5′ and 3′ untranslated regions,was inserted into the baculovirus transfer vector pAcG2T (Pharmingen) toproduce a GST fusion protein under the control of the strong AcNPVpolyhedrin promoter. This ILK transfer construct was then co-transfectedwith BaculoGold™ DNA (Pharmingen) into Sf9 insect cells (Invitrogen) anda high titre preparation of GST-ILK recombinant baculovirus was producedby amplification in Sf9 cells. Liter scale expression of GST-ILKrecombinant protein was done in 1000 mL spinner flasks (Bellco) byinfection of Hi5 insect cells (Invitrogen) grown in Ex-Cell™ 400 serumfree media (JRH Biosciences) at a multiplicity of infection ofapproximately 5. The cells were harvested three days after infection andlysed on ice in ILK Lysis Buffer (ILB; 10 mM imidazole, pH 7.5, 50 mMNaCl, 0.1% NP-40, 0.1% 8-mercaptoethanol, 0.5 mM PMSF, 1 mM benzamidine)with dounce tissue grinder (Kontes). The lysate was centrifuged at10,000 g for 15 minutes at 4° C. and the supernatant was discarded. Thepellet was re-suspended in ILB using the homogenizer and centrifuged asabove. Then the pellet was washed twice in ILK extraction buffer (IEB,10 mM imidazole, pH7.5, 400 mM NaCl, 1% NP-40, 0.1% β-mercaptoethanol,0.5 mM PMSF, 1 mM benzamidine). The pellet was then resuspended inDNAse-ATP buffer Buffer (DAB, 10 mM imidazole, PH7.5, 400 mM NaCl, 5 mMEDTA, 1% NP-40, 0.1% β-mercaptoethanol, 0.5 mM PMSF, 1 mM benzamidine,10 ug/mL DNAse I, 1 mM ATP, 10 mM MgCl₂, 1 mM MnCl₂, 5 uM β-methylaspartic acid, 2 mM NaF) and stirred for 30 minutes at ambienttemperature. The mixture was centrifuged at 10,000×g for 20 minutes andthe pellet resuspended and washed once in High Salt Buffer (HSB, 10 mMimidazole, PH7.5, 400 mM NaCl, 5 mM EDTA, 0.1% 8-mercaptoethanol, 0.5 mMPMSF, 1 mM benzamidine). The suspension was stirred for 30 minutes atambient temperature, and then centrifuged at 10,000 g for 20 minutes.Finally, the pellet was resuspended in ILK Storage Buffer (ISB, 10 mMimidazole, PH7.5, 0.2 mM EDTA, 0.1% β-mercaptoethanol, 0.5 mM PMSF, 30%glycerol) and stored at −80° C.

Biochemical analysis of the activated enzyme was performed onrecombinant human ILK1 protein preparation using the experimentalprotocol outlined in the section entitled “In Vitro Activity Profile ForKinases”. Typically, the ILK1 preparations were found to exhibit proteinphosphotransferase activity in the presence of 50 μM [γ-³²P]-ATP and 159μM ILK1 substrate (amino acid sequence: CKRRRLASLR-amide) during a 15minute reaction at ambient temperature.

Compounds were tested in the following assay for their ability toinhibit the activity of ILK. The desired in vitro potency of aparticular inhibitor is such that the compound is useful as atherapeutic agent, i.e. in the nanomolar or micromolar range. See Table1 infra.

A. Assay Description

Test compounds were lyophilized and stored at −20° C. Stock solutionswere made by weighing out the compounds and dissolving them in dimethylsulfoxide (DMSO) to a standard concentration, usually 20 mM, and storedat −20° C. The compounds were diluted to a starting intermediateconcentration of 250 μM in 1% DMSO, then serially diluted across a rowof a 96 well plate using serial 2 fold dilution steps. Diluted 100% DMSOwas used as a negative control.

5 μL of each compound dilution were robotically pipetted to Costar™serocluster plates maintaining the same plate layout. All assay mixturesconsisted of the following volumes:

-   -   5 μL diluted compound    -   10 μL target enzyme preparation    -   1 μL substrate    -   5 μL assay ATP

The assay mixtures were then incubated 15 minutes at ambienttemperature.

From each assay mixture, 10 μL of assay mixture was spotted ontoMillipore Multiscreen-PH™ opaque plates and washed twice for 10 minutesin 1% phosphoric acid. The plates were dried at 40° C. for 30 minutes,then substrate-phosphate complexes were quantitated by scintillationcounting. These Millipore plates are in a 96-well format withimmobilized P81 phosphocellulose membranes in the wells. Both thephosphorylated and non-phosphorylated form of the substrate bind to themembrane while ATP (unincorporated phosphate) is removed by subsequentwash steps.

B. Calculation of IC₅₀

Inhibition of ILK by the test compounds is measured by scintillationcounting of the incorporation of radioactive phosphate onto a specificsubstrate which is immobilized onto a filter paper at the end of theassay. To provide meaningful measurements of inhibition, the assays areperformed both in the absence and presence of specific and knowninhibitors, and the amount of incorporated radioactivity is compared toprovide a baseline measurement.

The “baseline activity” is the amount of radioactivity incorporated inthe absence of a target inhibitor. The amount of radioactivityincorporated in the presence of a target inhibitor is called the “sampleactivity”, and the % inhibition is expressed by the following formula:% inhibition=100−(sample activity/baseline activity*100)

and is usually expressed in conjunction with the compound concentration.By using a range of target inhibitor concentrations, the IC₅₀ of aninhibitor is estimated (i.e. the concentration at which enzymaticactivity is reduced by 50%). The IC₅₀ of various inhibitors against aparticular target can be compared, where a lower IC₅₀ indicates a morepotent inhibitor.

TABLE 1 Inhibition of ILK In Vitro Enzyme Assay IC₅₀ avg. Chemical Namevalue (μM)(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-[2-(1H-imidazol-4-yl)-ethyl]-amine0.2 (4-Benzothiazol-2-yl-2H-pyrazol-3-yl)-methyl-amine 1.7[2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazol-6-yl]- 0.1methanol[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-5-yl]-methanol 3.7[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-6-yl]-methanol 0.92-(1H-Pyrazol-4-yl)-benzothiazole 1.62-(1H-Pyrazol-4-yl)-benzothiazole-6-sulfonic acid amide 0.52-(3-Amino-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-6-sulfonic acid amide0.022-(3-Amino-5-methyl-1H-pyrazol-4-yl)-4,5,6-trifluoro-benzothiazole-7-0.07 sulfonic acid amide2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-6-carboxylic0.06 acid methyl ester2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-6-sulfonic0.04 acid methylamide2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-benzothiazole-6-carboxylic acid 1.12-(3-Amino-5-methyl-1H-pyrazol-4-yl)-benzothiazole-6-carboxylic acid 0.3methyl ester2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 0.07methylamide2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid (2,6-2.6 dimethyl-pyrimidin-4-yl)-amide2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-benzothiazole-7-carboxylic acid 0.7methyl ester 2-(3-Methyl-1H-pyrazol-4-yl)-benzothiazole 0.92-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-phenol 4.62-(5-Amino-1H-pyrazol-4-yl)-benzothiazol-6-ylamine 0.52-(5-Amino-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid methylamide0.062-(5-Amino-3-methyl-1H-pyrazol-4-yl)-4-fluorobenzothiazole-6-sulfonic0.04 acid amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-4-sulfonic0.15 acid (2-hydroxy-ethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-6-sulfonic0.007 acid amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-6-sulfonic0.05 acid (2-hydroxy-ethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-6-sulfonic0.13 acid (pyridin-4-ylmethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-5-ol 0.22-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-5-sulfonic acid 1.9methylamide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-carboxylic acid 0.3amide 2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid0.01 amide 2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonicacid (2- 0.2 hydroxy-ethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid (2-0.6 methoxy-ethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 4-4.8 fluoro-benzylamide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid (2-1.7 thiophen-2-yl-ethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 4-0.9 chloro-benzylamide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 4-1.0 methoxy-benzylamide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 0.8benzylamide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 0.8phenethyl-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid[2-(4- 0.2 amino-phenyl)-ethyl]-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid (2-3.0 morpholin-4-yl-ethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 0.09(2,2,2-trifluoro-ethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 0.7cyclopropylmethyl-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid [2-0.2 (3H-imidazol-4-yl)-ethyl]-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 4-0.6 amino-benzylamide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 0.04(pyridin-4-ylmethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid (2-0.3 dimethylamino-ethyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid (3-3.2 dimethylamino-propyl)-amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 0.03(acetic hydrazido) amide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid 0.9(phenylhydrazino) amide2-(5-Amino-3-pyridin-4-yl-1H-pyrazol-4-yl)-benzothiazole-6-sulfonic acid0.05 amide 2-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-ethanol0.4 2-(5-Amino-4-benzothiazol-2-yl-2H-pyrazol-3-ylamino)-cyclopentanol3.5 2-{[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-6-ylmethyl]-0.1 amino}-ethanol3-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-propan-1-ol 0.23-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-propan-1-ol 0.43-[5-Amino-4-(5-fluoro-6-methoxy-benzothiazol-2-yl)-1H-pyrazol-3- 0.5ylamino]-propan-1-ol3-{[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-6-ylmethyl]- 0.8amino}-benzenesulfonamide4-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-6-ethyl-benzene-1,3-diol 1.94-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-benzene-1,3-diol 3.34-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-phenol 3.44-(4-fluorobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine 0.44-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-butan-1-ol 1.04-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-butan-1-ol 1.74-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-butyric acid 4.34-(5-Amino-4-benzothiazol-2-yl-2H-pyrazol-3-ylamino)-N-thiazol-2-yl- 3.5benzenesulfonamide4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-(2-fluoro-phenyl)-2H-pyrazol-4.0 3-ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-(2-phenyl-cyclopropyl)-2H-2.1 pyrazol-3-ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-(3-fluoro-phenyl)-2H-pyrazol-2.3 3-ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-(3-nitro-phenyl)-2H-pyrazol-0.9 3-ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-(4-fluoro-phenyl)-2H-pyrazol-2.4 3-ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-(4-methoxy-phenyl)-2H- 2.0pyrazol-3-ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-(5-nitro-furan-2-yl)-2H- 0.3pyrazol-3-ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-furan-2-yl-2H-pyrazol-3- 0.04ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-isoxazol-5-yl-2H-pyrazol-3-0.1 ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine0.064-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-phenyl-2H-pyrazol-3-ylamine2.34-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-piperazin-1-yl-2H-pyrazol-3-0.07 ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-pyridin-4-yl-2H-pyrazol-3-0.2 ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-thiophen-2-yl-2H-pyrazol-3-0.7 ylamine4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-N3-[2-(3H-imidazol-4-yl)-ethyl]-0.2 1H-pyrazole-3,5-diamine4-(5-Fluoro-6-methyl-benzothiazol-2-yl)-2H-pyrazol-3-ylamine 0.054-(5-Fluoro-6-methyl-benzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine0.3 4-(5-Fluoro-benzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine 0.044-(5-Methoxy-benzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine 3.74-(5-Trifluoromethyl-benzothiazol-2-yl)-1H-pyrazol-3-ylamine 1.14-(6-Bromo-5-fluoro-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine 0.24-(6-Bromo-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine 0.44-(6-Chlorobenzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine 0.34-(6-Dimethylaminomethyl-5-fluoro-benzothiazol-2-yl)-5-methyl-1H- 0.6pyrazol-3-ylamine4-(6-Dimethylaminomethyl-benzothiazol-2-yl)-5-methyl-2H-pyrazol-3- 0.2ylamine 4-(6-Fluoro-benzothiazol-2-yl)-1H-pyrazol-3-ylamine 0.24-(6-Fluoro-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine 0.44-(6-Methanesulfonyl-benzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine0.5 4-(6-Methoxy-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-ylamine 0.344-(6-Methoxy-benzothiazol-2-yl)-5-piperazin-1-yl-2H-pyrazol-3-ylamine0.2 4-(6-Nitro-benzothiazol-2-yl)-2H-pyrazol-3-ylamine 3.44-(7-chloro-4-methoxy-benzothiazol-2-yl)-5-methyl-2H-pyrazol-3-ylamine1.04-(7-Chloro-5-fluoro-6-methoxy-benzothiazol-2-yl)-5-methyl-1H-pyrazol-0.4 3-ylamine4-[(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-methyl]- 2.9benzenesulfonamide4-[2-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-ethyl]-phenol2.9 4-Benzothiazol-2-yl-1H-pyrazol-3-ylamine 0.34-Benzothiazol-2-yl-1H-pyrazole-3,5-diamine 0.74-Benzothiazol-2-yl-5-(3-dimethylamino-propyl)-2H-pyrazol-3-ylamine 0.24-Benzothiazol-2-yl-5-(3-methylamino-propyl)-2H-pyrazol-3-ylamine 0.024-Benzothiazol-2-yl-5-(4-dimethylamino-butyl)-2H-pyrazol-3-ylamine 0.44-Benzothiazol-2-yl-5-(4-methylamino-butyl)-2H-pyrazol-3-ylamine 0.14-Benzothiazol-2-yl-5-(4-nitro-phenyl)-2H-pyrazol-3-ylamine 4.04-Benzothiazol-2-yl-5-cyclopropyl-2H-pyrazol-3-ylamine 0.64-Benzothiazol-2-yl-5-ethyl-1H-pyrazol-3-ylamine 0.84-Benzothiazol-2-yl-5-furan-2-yl-2H-pyrazol-3-ylamine 0.14-Benzothiazol-2-yl-5-methyl-1H-pyrazol-3-ylamine 0.34-Benzothiazol-2-yl-5-methylsulfanyl-1H-pyrazol-3-ylamine 1.14-Benzothiazol-2-yl-5-phenyl-1H-pyrazol-3-ylamine 0.94-Benzothiazol-2-yl-5-piperazin-1-yl-2H-pyrazol-3-ylamine 0.194-Benzothiazol-2-yl-5-piperidin-4-yl-2H-pyrazol-3-ylamine 0.064-Benzothiazol-2-yl-5-pyridin-3-yl-2H-pyrazol-3-ylamine 0.64-Benzothiazol-2-yl-5-pyridin-4-yl-2H-pyrazol-3-ylamine 0.14-Benzothiazol-2-yl-5-pyrrolidin-1-yl-1H-pyrazol-3-ylamine 3.74-Benzothiazol-2-yl-5-thiophen-2-yl-2H-pyrazol-3-ylamine 0.94-Benzothiazol-2-yl-N3-(1H-imidazol-2-ylmethyl)-1H-pyrazole-3,5- 2.5diamine4-Benzothiazol-2-yl-N3-(1H-imidazol-2-ylmethylene)-1H-pyrazole-3,5- 1.0diamine4-Benzothiazol-2-yl-N3-(2-dimethylamino-ethyl)-1H-pyrazole-3,5-diamine0.5 4-Benzothiazol-2-yl-N3-(2-ethylamino-ethyl)-1H-pyrazole-3,5-diamine0.15 4-Benzothiazol-2-yl-N3-(2-methoxy-ethyl)-1H-pyrazole-3,5-diamine0.74-Benzothiazol-2-yl-N3-(2-pyrrolidin-1-yl-ethyl)-1H-pyrazole-3,5-diamine1.9 4-Benzothiazol-2-yl-N3-(3-dimethylamino-propyl)-1H-pyrazole-3,5- 0.3diamine4-Benzothiazol-2-yl-N3-(3-imidazol-1-yl-propyl)-1H-pyrazole-3,5-diamine0.54-Benzothiazol-2-yl-N3-[2-(1H-indol-3-yl)-ethyl]-1H-pyrazole-3,5-diamine3.3 4-Benzothiazol-2-yl-N3-piperidin-4-ylmethyl-1H-pyrazole-3,5-diamine2.74-Benzothiazol-2-yl-N5-(2-morpholin-4-yl-ethyl)-1H-pyrazole-3,5-diamine1.5 4-Benzothiazol-2-yl-N5-ethyl-1H-pyrazole-3,5-diamine 1.74-Benzothiazol-2-yl-N5-pyridin-3-ylmethyl-1H-pyrazole-3,5-diamine 2.95-(2-Chloro-pyridin-3-yl)-4-(5-fluoro-6-methoxy-benzothiazol-2-yl)-2H-3.4 pyrazol-3-ylamine5-(3-Amino-propyl)-4-benzothiazol-2-yl-2H-pyrazol-3-ylamine 0.015-(4-Amino-phenyl)-4-benzothiazol-2-yl-2H-pyrazol-3-ylamine 0.15-Cyclopropyl-4-(5-fluoro-6-methoxy-benzothiazol-2-yl)-2H-pyrazol-3- 1.0ylamine5-Methyl-4-(4,5,6-trifluoro-benzothiazol-2-yl)-1H-pyrazol-3-ylamine 0.25-Methyl-4-(5-trifluoromethylbenzothiazol-2-yl)-1H-pyrazol-3-ylamine 2.85-Methyl-4-(6-methylaminomethyl-benzothiazol-2-yl)-2H-pyrazol-3- 0.5ylamine5-Methyl-4-(6-morpholin-4-ylmethyl-benzothiazol-2-yl)-2H-pyrazol-3- 0.5ylamine5-Methyl-4-(6-pyrrolidin-1-ylmethyl-benzothiazol-2-yl)-2H-pyrazol-3- 0.9ylamine5-Methyl-4-[6-(4-methyl-piperazin-1-ylmethyl)-benzothiazol-2-yl]-2H- 5.0pyrazol-3-ylamine5-Methyl-4-[6-(4-methyl-piperazine-1-sulfonyl)-benzothiazol-2-yl]-2H-4.9 pyrazol-3-ylamineN-[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazol-6-yl]-acetamide 0.4N-[2-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-ylamino)-ethyl]- 1.1acetamide N-[4-(5-Amino-4-benzothiazol-2-yl-1H-pyrazol-3-yl)-phenyl]-1.6 hydroxylamineN-{2-[2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole-6- 0.7sulfonylamino]-ethyl}-acetamideN-{2-[5-Amino-4-(5-fluoro-6-methoxy-benzothiazol-2-yl)-1H-pyrazol-3- 1.8ylamino]-ethyl}-acetamideN3-(2-Amino-ethyl)-4-benzothiazol-2-yl-1H-pyrazole-3,5-diamine 0.02N3-(2-Dimethylamino-ethyl)-4-(5-fluoro-6-methoxy-benzothiazol-2-yl)-1H-0.3 pyrazole-3,5-diamineN3-(3-Dimethylamino-propyl)-4-(5-fluoro-6-methoxy-benzothiazol-2-yl)-0.2 1H-pyrazole-3,5-diamineN3-(4-Amino-phenyl)-4-benzothiazol-2-yl-1H-pyrazole-3,5-diamine 5.0N3-[2-(3H-Imidazol-4-yl)-ethyl]-4-(6-methoxy-benzothiazol-2-yl)-1H- 0.6pyrazole-3,5-diamine

Example 40 Cytotoxicity Assay

This procedure was used to assess the effects compounds have on variouscell lines with respect to cell viability. Cell viability is quantifiedusing calcein AM and measuring its conversion to a fluorescent product(calcein) with a fluorimeter.

The principle of this assay is based on the presence of ubiquitousintracellular esterase activity found in live cells. By enzymaticreaction of esterase, non-fluorescent cell-permeant calcein AM isconverted to the intensely fluorescent calcein. The polyanionic dyecalcein is retained within live cells, producing a green fluorescence inlive cells. It should be noted that calcein AM is susceptible tohydrolysis when exposed to moisture. Therefore, prepare aqueous workingsolutions containing calcein AM immediately prior to use, and use withinabout one day.

A kit available to do this assay is “LIVE/DEAD® Viability/CytotoxicityKit (L-3224)” by Molecular Probes.

Cells were collected from tissue culture flasks and trypsinized,centrifuged, resuspended and counted. Cells were seeded to obtain 80-90%confluence (for normal cells, 10,000 cells/well (8000 cells/well forHUVEC cells)). A cell concentration of 110,000 cells/mL (88,000cells/well for HUVEC cells) is prepared as 90 μL volume is used perwell.

Using an 8-channel multi-dispense pipettor, cells were seeded in thecentral rows of the plate (Nunclon™ 96 well flat-bottom plate), leavingthe peripheral top and bottom rows with same volume of media only. Theplates were incubated at 37° C., 5% CO₂ overnight for approximately 24hours.

For test compounds, cell culture media (e.g., RPMI+10% FBS), 10×compound solution of final desired concentration from 20 mM stockcompounds was prepared. Ten μl of this 10× compound solution is added tothe 90 μL of cells already present in the 96 well plates and a knowncytotoxic compound from previous testing is used as a positive control.The negative control is 100% DMSO diluted to the same factor as thecompounds.

The plates are incubated at 37° C. for approximately 24 hours, and mediais aspirated after plates are spun at 2400 rpm for 10 min at ambienttemperature. 100 μL of 1×DPBS (without CaCl₂, without MgCl₂ (GibcoBRL,cat#14190-144)) is added to each well.

The calcein AM solution is prepared by adding 50 μg of calcein AMcrystal (m.w.=994.87 g/mol, Molecular Probes, Eugene, Oreg.) andanhydrous DMSO (Sigma Aldrich) to make 1 mM stock and diluting stock to2× the final desired concentration in 1×DPBS just before the assay. 100μL is then added to the 100 μL of DPBS in the wells, and the plates areincubated at ambient temperature for 30 minutes. Fluorescence data wasread and recorded (Fluoroskan Ascent® FL fluorimeter (excitation˜485 nm,emission˜527 nm)).

The values for replicates (usually six) are averaged and % inhibition iscalculated as follows:% inhibition=100−[(AVG treatment−AVG positive control)/(AVG negativecontrol−AVG positive control)*100]

On cell lines HUVEC, HS27, and LL-86, cytotoxicity of 62 representativecompounds in Table 1, at 5 μM and 25 μM concentration, ranged from zeropercent to 20%. Most of the measurements were less than 10%.

Example 41 Cell Invasion in Matrigel™ Extracellular Matrix Extract

This procedure is used to assess the compound effect on the tumor cellinvasion through Matrigel™-coated Fluoroblok™ inserts. Invasion allowstumor cells to spread to sites other that the primary tumor. Thefollowing assay uses this system to assess compound effects on theanti-tumor cell invasion through layer of Matrigel™ extracellularmatrix.

The cell lines used are HT 1080 (ATCC, Cat# CCL-121), DU-145 (ATCC, Cat#HTB-81), PC3 (ATCC, Cat# CRL-1435) or B16F1 (ATCC, Cat# CRL-6323).

The invasion test system (BD Bioscience's BioCoat™ FluoroBlok™ InvasionSystem including BD BioCoat™ Matrigel™ Invasion Chambers with thefluorescence blocking membrane FluoroBlok™ 24-Multiwell Insert System™)is removed from the package from −20° C. storage and allowed to warm toambient temperature. PBS is added to the interior of the inserts andthey are allowed to rehydrate for 2 hours at 37° C. The medium is thenremoved and 450 μL cell suspensions of tumor cells (grown to 50-70%confluence, trypsinized, and resuspended in medium without serum at1×10⁶/mL) is added to the top chamber. Test compounds are added to thetop chamber at 10× the desired final concentration in 50 μL volumes.DMSO acts as the control.

Then 750 μL of medium containing 50% fresh growth medium with 10% FBSand 50% NIH 3T3-conditioned medium is added to each of the bottom wells.The invasion system is incubated for 24 to 48 hours at 37° C., in a 5%CO₂ atmosphere.

Following incubation, the insert plate is transferred into a second24-well plate containing 0.5 mL of 5 μg/mL calcein AM in Hanks bufferedsalt solution (HBSS), and plates are incubated for 1 hour at 37° C., 5%CO₂.

Fluorescence data indicating cell invasion is read in a FluoroskanAscent™ FL (LabSystems) with bottom reading at excitation/emissionwavelength of 485/538 nm.

Data is expressed as fluorescence units (FU) from the sum of middle 25areas per 24-well or as percentage of invasion inhibition by followingformula: % of invasion inhibition=100−FU of compound treated cellinvasion/FU of DMSO treated cell invasion*100.

Twenty-one representative compounds from Table 1 were tested in thisassay, and the percent of inhibition ranged from 20 to 80%. Thecompounds are thus useful to prevent metastasis in cancer and tissueremodeling.

Example 42 Inflammatory Responses are Modulated in the Presence ofCompounds

Establishment of Inflammation Assay Panel.

Macrophages are important elements of innate immunity to infection andare among the first cell type in the immune response to be exposed toand activated by infectious agents. IFN-γ and LPS are potent activatorsof macrophages, priming them for a variety of biological effects. IFN-γ,initially secreted by NK and T cells in response to infection, convertsmacrophages from a resting to an activated state, priming them forantimicrobial activity manifested by increased killing of intracellularpathogens, and antigen processing and presentation to lymphocytes. Theaction of IFN-γ is synergized with the LPS second messenger, enhancingthe stimulation of macrophages through the activation of NF-κB, thatresults in the transcriptional up-regulation of a number of genesinvolved in the cell-mediated immune response, including induciblenitric oxide synthase (iNOS). Activated macrophages are qualitativelydifferent from quiescent macrophages. These differences are typicallyobserved by an increased proliferation index, up-regulated expression ofMHC-II, and production of various bioactive molecules. The latterbiological effects are mediated by nitric oxide (NO) release andincreased production of pro-inflammatory cytokines (IL-6, TNF-α, IL-1).Primary macrophages derived from Balb/c and RAW 264.7 cells (Balb/cbackground) were used to establish in vitro inflammatory models withfast and reliable readouts.

Materials and Methods

1. Reagents.

The iNOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and murine rIFN-γwere purchased from Calbiochem (San Diego, Calif.). Protein-free,phenol/water-extracted LPS (from E. coli serotype 0111:B4 0127:B8),Zymosan A, dexamethasone and hydrocortisone, sulfanilamide andN-(1-naphthyl)-ethylenediamine, were purchased from Sigma (St. Louis,Mo.). Human recombinant vascular endothelial growth factor (VEGF) waspurchased from R&D Systems (Minneapolis, Minn.). Anti-murine iNOS/NOStype II antibodies were obtained from Transduction Laboratories(Lexington, Ky.). Female, 6-12 wk of age, BALB/c mice were purchasedfrom Harlan Inc. (Indianapolis, Ind.) and maintained in compliance withthe Canadian Council on Animal Care standards.

2. Isolation of Primary Mouse Macrophages.

Peritoneal exudate macrophages were isolated by peritoneal lavage withice-cold sterile physiological saline 24 hours after intraperitonealinjection of BALB/c mice with 0.5 mL of sterile Zymosan A (1 mg/0.5 mL0.9% saline). Cells were washed, resuspended in RPMI 1640 supplementedwith 1 mM D-glucose, 1 mM sodium pyruvate, 100 units/mL penicillin, 100μg/mL streptomycin, and 5% FBS.

3. Inhibition of IL-12 Release Determination

Murine primary macrophage are activated following incubation with LPS inthe presence of sub-optimal doses of IFN-γ. Upon activation, macrophagesparticipate actively in the onset of inflammation by releasing bioactivemolecules that amplify the initial inflammatory response. Stimulatedmacrophages demonstrate up-regulated expression of MHC-II receptors,increased release of NO and produce a number of pro-inflammatorycytokines including IL-12, IL-6, TNF-α, MIP-1α and MIP-1β.

Briefly, IL-12 levels in the supernatants from stimulated macrophageswere determined with PharMingen's OptEIA™ ELISA set developed using ananti-mouse IL-12 antibody pair and mouse rIL-12 standard (PharMingen).Maxisorp™ F16 multiwell strips (Nunc, Roskilde, Denmark) were coatedwith anti-mouse IL-12 capture Ab (at recommended concentration) in 0.1 MNaHCO₃, pH 9.5, 100 μL/well, overnight at 4° C. Plates were washed 3×with 0.05% Tween 20 in PBS (PBST) and blocked for 1 h with 200 mL/wellof 10% FCS in PBS (blocking and dilution buffer). Plates were washed 3×with PBST and duplicate samples (100 μL/well) or standards (100 μL/well)in diluent buffer were incubated for 2 h. Plates were washed five timeswith PBST and incubated with biotinylated anti-mouse IL-12 andavidin-horseradish peroxidase (HRP) conjugate (at concentrationsrecommended by the manufacturer) for 1 h. Plates were washed 7× withPBST and 100 μl of 3,3′,5,5′-tetramethylbenzidine substrate solution wasadded to each well. After 15-30 minute incubation at room temperature,colour development was terminated by adding 50 μL of 2 NH₂SO₄.Absorbance was read at 450 nm with an EL 312e microplate reader. Thedetection limit for IL-12 was 15.6 pg/ml.

For discussion of measuring Interleukin-12 in tissue culturesupernatants, see, e.g., Skeen M. J., Miller M. A., Shinnick T. M., etal. J. Immunol. (1996) 156(3):1196-206. Results for the IL-12 study areshown in Table 2 for representative compounds.

4. Inhibition of TNFα Release Determination:

Murine primary macrophage will get activated following incubation withLPS in the presence of sub-optimal doses of IFN-γ. Upon activation,macrophages participate actively in the onset of inflammation byreleasing bioactive molecules that amplify the initial inflammatoryresponse Activated macrophages demonstrate up-regulated expression ofMHC-II receptors, increased release of NO and produce a number ofpro-inflammatory cytokines including TNF-α.

TNF-αlevels in the supernatants from stimulated macrophages weredetermined with PharMingen's OptEIA TNF-α kit (PharMingen). Theexperiment was performed at room temperature, about 21° C., unlessotherwise stated.

The microwells (F8 MaxiSorp™ Loose/Nunc-Immuno Module; Gibco/BRL) werecoated with 100 μl/well of capture antibody (at the recommendedconcentration) and incubated overnight at 4° C. The plates were washedwith 100 μl/well of wash buffer (0.05% Tween-20 in 1×PBS) and blockedfor 1 hour with 200 μl/well assay diluent buffer (10% FBS in 1×PBS).Next, the solution was removed and the plates were washed 5× with washbuffer.

The TNF-αanalysis was initiated by adding duplicate samples (100μl/well) or standards (100 μl/well) in diluent buffer which wereincubated for 2 h. The solution was removed and the plates washed 5×with wash buffer. Enzyme reagent (100 μl) containing biotinylated mouseTNF-αmonoclonal antibody and avidin-horseradish peroxidase conjugate (atconcentrations recommended by the manufacturer) was added to each well.The plate was incubated for 1 hour, the solution removed, and the plateswashed 7× with wash buffer. Finally, 3,3′,5,5′-tetramethylbenzidine(TMB) substrate solution (100 μl) was added to each well and the plateincubated in the dark for 15 to 30 minutes. Colour development wasterminated by adding 50 μl of 2N H₂SO₄. The optical density was measuredat 450 nm with an EL 312e microplate reader. The detection limit forTNF-α was 15.6 pg/ml.

The data is represented as a percentage of TNF-αinhibition by thefollowing formula: % TNF-αinhibition=100−(AVG treatment/AVG DMSOcontrol)*100.

For discussion of measuring Tumour Necrosis Factor (TNF-αtissue culturesupernatants, see e.g. Drew, P. D., and J. Chavis, “Inhibition ofMicroglial Cell Activation by Cortisol” Brain Research Bulletin. (2000)52(5):391-396; Drew, P. D., and J. Chavis “Female Sex Steroids: EffectsUpon Microglial Cell Activation” J. Neuroimmunology. (2000)111(1-2):77-85.

Representative compounds were tested in this assay, and the results areshown in Table 2.

5. Inhibition of IFNγ Release Determination:

T-lymphocytes obtained from mice spleens are a suitable sample forstudying the activation properties of this immunologically importantcell type. T-lymphocytes are the main regulators for inflammation.Concanavalin A (ConA) is an effective activator of T-lymphocytes, whichallows T-lymphocytes to proliferate and produce inflammatory cytokines,such as Interferon (IFNγ) and regulatory cytokines, such as Interleukin10 (IL-10).

Isolation of T-Lymphocytes from Mouse Spleens

Spleens were removed from Balb/c mice place in 3 ml serum-free RPMI 1640(Gibco/BRL) and stored on ice until ready for use. The spleens weretransferred onto mesh screen containing 10 ml of ice cold RPMI-5 (1×sodium pyruvate and 5% FBS) and gently ground with a pestle. The cellsuspensions were centrifuged at 1500 rpm for 6 minutes at 4° C. The redblood cells were lysed by adding 2 ml of lysis buffer for 1 minute andthe reaction terminated quickly by adding 10 ml of RPMI-5. Thesupernatant was discarded and the pellet washed two additional times.The cells were resuspended in RPMI-5 and the cell suspensions combined.The cells were counted and their concentration was adjusted to 2.8×10⁶cells/ml using RPMI-5.

Stimulation and Treatment of Splenocytes

The isolated splenocytes (180 μl/ml of 2.8×10⁶ cells/ml) were added to96-well plates for a final concentration of 5×10⁵ cell/well. The workingconcentration of ConA was 2.5 μl/ml. A 20× working concentration ofConcanavalin A was 50 μl/ml was prepared using RPMI-5. The testcompounds, dimethylsulfoxide (DMSO) negative vehicle control andstaurosporine positive control were diluted twenty times using RPMI-5.Ten μL each of DMSO and staurosporine were combined with 180 μl ofmedium containing splenocytes in the wells, and 10 μl of 50 μg/ml ConAwas immediately added. Medium without ConA was added to the controlwells. To test the compounds on unstimulated splenocytes, 10 μl ofRPMI-5 was added to each well in place of the ConA solution. The plateswere incubated at 37° C. in 5% CO₂ in humidified conditions overnight(18-24 hours) for INFγ.

Enzyme Immunoassays for Mouse INFγ

At the completion of the stimulation described above, the plates werespun at 2000 rpm for 10 minutes. The supernatants (100 μl) from eachwell were transferred to fresh plates for INFγ analysis by ELISA.

INFγ levels in the supernatants from stimulated lymphocytes weredetermined with PharMingen's OptEIA™ INFγ Kit (PharMingen) according tomanufacturer's directions. The data, shown in Table 2 infra, isrepresented as a percentage of INFγ inhibition by the following formula:INFγ inhibition=100−(AVG treatment/AVG DMSO control)*100.

6. Measuring Cytotoxicity by MTS Staining

An assessment of cell viability subsequent to compound exposure wasdetermined quantitatively by employing a cytotoxic assay using thesoluble tetrazolium salt[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] (MTS). The MTS solution was made fresh and under low light.The electron coupling reagent phenazine methosulfate (PMS) was added atthe time of the assay. Twenty μL of MTS solution per 100 μl of tissueculture medium was added to each well. The plates were incubated 37° C.for 4 hours for unstimulated and 18-24 ConA stimulated splenocytes. Theabsorbance was read at 490 nm wavelength. A kit available to do thisassay is “CellTiter 96® AQ Kit AQ_(ueous) Kit (G5421)” by Promega.

The values for four replicates are averaged and % inhibition iscalculated at follows: % survival=100−[(AVG treatment−AVG positivecontrol)/(AVG negative control−AVG positive control)*100]. Results areshown in Table 2.

For discussion of measuring INFγ in tissue culture supernatants, seee.g., Uzonna, J., Kaushik, R., Gordon, J., and Tabel, H. J. Immuno.(1998), 161:5507-5515; Xi S., Cohen D., and Chen L. J. of LipidResearch. (1998), 39, 1677-1687. Product Information, “LIVE/DEAD®Viability/Cytotoxicity Kit (L-3224)” by Molecular Probes (revised 24Jan. 2001).

7. Inhibition of MCP-1 Release

The activation of endothelial cells by pro-inflammatory cytokine TNF-αleads to the production of several chemokines. The release of thesechemokines play an important role in leukocyte trafficking andextravasation of leukocytes into tissue during inflammation. MCP-1 andIL-8 are among the chemokines released upon stimulation of humanendothelial cells (HUVECs) by TNFα, and contribute to migration ofmonocytes to the sites of inflammation. Cells should not be passagedmore than 6 times.

Cells were maintained and grown in tissue culture flasks (T75) inendothelial cell growth medium (EGM, Clonetics) containing 5 additionalsupplements (EGM-5) in medium [human recombinant endothelial growthfactor (hEGF), hydrocortisone (HC), bovine brain extract (BBE), fetalbovine serum (FBS) and Gentamycin (GA)] incubated at 37° C., 5% CO₂. Thecells should be propagated a maximum of 20 passages.

Cells were trypsinized (0.25%), collected from the tissue cultureflasks, centrifuged, resuspended in EGM-5, and counted. The cellconcentration was adjusted to 2.2×10⁴ cells/ml with EGM-5 medium. TheHUVEC were seeded at 180 μl of 2.2×10⁴ cells/ml into 96-well plates fora final seeding density of 4,000 cells/well. The plates were incubatedovernight at 37° C., 5% CO₂ to enable the cells to attach and grow.

The working concentration of TNFα was 1,000 pg/ml. A twenty timesworking concentration of TNFα was 20,000 pg/ml was prepared using EGM-5.The test compounds, DMSO negative vehicle control and staurosporinepositive control were diluted 20× using EGM-5. Ten μL each of thecompounds, DMSO and staurosporine were combined with 180 μl of mediumcontaining HUVEC cells. Immediately following the addition of thecompounds, 10 μl of 50 μg/ml TNFα was added to the wells. Medium withoutTNFα was added to the control wells. To test the compounds onunstimulated HUVEC cells, 10 μl of EGM-5 was added to each well in placeof the TNFα solution. The plates were incubated at 37° C. in 5% CO₂ inhumidified conditions overnight (18-24 hours) for TNFα.

At the completion of the stimulation, the plates were spun at 2000 rpmfor 10 minutes. The supernatants from each well were transferred tofresh plates for MCP1 analysis by ELISA.

TNFα levels in the supernatants from stimulated lymphocytes weredetermined with PharMingen's OptEIA™ MCP1 Kit (PharMingen) according tomanufacturer's instructions. The optical density was measured at 450 nmwith an EL 312e microplate reader. The detection limit for MCP1 was 15.6pg/ml.

The data is represented as a percentage of MCP1 inhibition by thefollowing formula: % MCP1 inhibition=100−(AVG treatment/AVG DMSOcontrol)*100. Results are shown in Table 2.

For discussion of measuring MCP1 in tissue culture supernatants, seee.g., Kalogeris T. J., Laroux F. S., Cockrell A. et al. Am J. Physiol.276 (4 Pt 1):C856-864; Instructions provided by PharMingen OptEIA humanMCP-1 set (PharMingen, Cat#555179).

Measuring Cytotoxicity by MTS Staining was Performed as Above.

The values of replicates are averaged and % inhibition is calculated asfollows: survival=100−[(AVG treatment−AVG positive control)/(AVGnegative control−AVG positive control)*100].

Results are shown in Table 2.

TABLE 2 IC₅₀ of Compounds on Stimulated Macrophages and Splenocytes IFNgon ConA 1° Survival TNFa on IL-12 on spleno- of ConA macro- macro-Survival MCP1 on cytes 1° spleno- phages phages of macro- HUVECSChemical Name (μM) cytes (μM) (μM) phages (μm) (4-Benzothiazol-2-yl-1H-8.4 uM >25 11.76 uM 8.1 uM 14.5 uM 21.3 uM pyrazol-3-yl)-[2-(1H-imidazol-4-yl)-ethyl]-amine [2-(3-Amino-5-methyl-1H- 2.026 >25pyrazol-4-yl)-5-fluoro- benzothiazol-6-yl]-methanol 2-(1H-Pyrazol-4-yl)-10.3 uM >25 benzothiazole-6-sulfonic acid amide 2-(3-Amino-1H-pyrazol-4-2.126 >25 yl)-5-fluoro-benzothiazole-6- sulfonic acid amide2-(3-Amino-5-methyl-1H- 1.9 uM 10.2 uM pyrazol-4-yl)-4,5,6-trifluoro-benzothiazole-7-sulfonic acid amide 2-(3-Amino-5-methyl-1H- 1.03 >25pyrazol-4-yl)-5-fluoro- benzothiazole-6-carboxylic acid methyl ester2-(3-Amino-5-methyl-1H- .368 uM 11.6 uM pyrazol-4-yl)-5-fluoro-benzothiazole-6-sulfonic acid methylamide 2-(3-Amino-5-methyl-1H- 0.8 uM23.0 1.5 1.6 6.4 4.2 pyrazol-4-yl)-benzothiazole- 6-sulfonic acidmethylamide 2-(3-Methyl-1H-pyrazol-4- 21.4 uM N >25 12.4 >25 >25yl)-benzothiazole 2-(5-Amino-1H-pyrazol-4- 2.461 >25yl)-benzothiazole-6-sulfonic acid methylamide 2-(5-Amino-3-methyl-1H-3.2 uM >25 6.6 uM 11.7 uM >25 >25 pyrazol-4-yl)-4-fluorobenzothiazole-6- sulfonic acid amide 2-(5-Amino-3-methyl-1H- 8.9uM >25 5.204 uM 12.4 uM >25 >25 pyrazol-4-yl)-5-fluoro-benzothiazole-4-sulfonic acid (2-hydroxy-ethyl)-amide2-(5-Amino-3-methyl-1H- 1 uM 15.1 uM .9 uM 2.7 uM 6.7 uM 2.7 uMpyrazol-4-yl)-5-fluoro- benzothiazole-6-sulfonic acid amide2-(5-Amino-3-methyl-1H- 3.4 uM >25 2.0 uM 4.8 uM 14.597 9.0 uMpyrazol-4-yl)-5-fluoro- benzothiazole-6-sulfonic acid(2-hydroxy-ethyl)-amide 2-(5-Amino-3-methyl-1H- 3.3 uM 14.8 uM 4.4 uM6.9 uM 23.4 uM 11.7 uM pyrazol-4-yl)-5-fluoro- benzothiazole-6-sulfonicacid (pyridin-4-ylmethyl)- amide 2-(5-Amino-3-methyl-1H- 11.2 uM >25 7.6uM 5.5 uM >25 N pyrazol-4-yl)-benzothiazol- 5-ol 2-(5-Amino-3-methyl-1H-4.7 uM >25 8.0 uM 9.6 uM >25 16.2 uM pyrazol-4-yl)-benzothiazole-6-carboxylic acid amide 2-(5-Amino-3-methyl-1H- 6.9 uM N 10.6 uM >2514.2 uM pyrazol-4-yl)-benzothiazole- 6-sulfonic acid (2-methoxy-ethyl)-amide 2-(5-Amino-3-methyl-1H- 5.4 uM >25 21.7 uM 7.8 uM >25 14.5uM pyrazol-4-yl)-benzothiazole- 6-sulfonic acid [2-(4-amino-phenyl)-ethyl]-amide 2-(5-Amino-3-methyl-1H- 1.5 uM 3.8 uM 3.3 uM 2.1 uM9.7 uM 6.3 uM pyrazol-4-yl)-benzothiazole- 6-sulfonic acid (pyridin-4-ylmethyl)-amide 2-(5-Amino-3-methyl-1H- 2.8 uM >25 3.9 3.4 7.0 12.4pyrazol-4-yl)-benzothiazole- 6-sulfonic acid amide2-(5-Amino-3-methyl-1H- 3.7 uM 23.1 uM 5.6 uM 3.0 uM 14.7 uM 5.9 uMpyrazol-4-yl)-benzothiazole- 6-sulfonic acid (2,2,2-trifluoro-ethyl)-amide 2-(5-Amino-3-methyl-1H- 3.5 uM >25 6.13 uM 5.8 uM14.8 uM 11.9 uM pyrazol-4-yl)-benzothiazole- 6-sulfonic acid (2-hydroxy-ethyl)-amide 2-(5-Amino-3-methyl-1H- 10.8 uM >25 7.9 uM 5.1 uM >25 14.9uM pyrazol-4-yl)-benzothiazole- 6-sulfonic acid (2-dimethylamino-ethyl)-amide 2-(5-Amino-3-pyridin-4-yl- 11.7 uM >25 13.2uM 17.7 uM >25 >25 1H-pyrazol-4-yl)- benzothiazole-6-sulfonic acid amide2-{[2-(5-Amino-3-methyl- 8.1 uM 20.1 uM 1H-pyrazol-4-yl)-benzothiazol-6-ylmethyl]- amino}ethanol 3-(5-Amino-4-benzothiazol- 10.3uM N >25 14.0 uM N ~25 2-yl-1H-pyrazol-3-yl)- propan-1-ol3-(5-Amino-4-benzothiazol- 9.4 uM >25 18.2 uM 10.6 uM >25 17.3 uM2-yl-1H-pyrazol-3-ylamino)- propan-1-ol 4-(5-Fluoro-6-methoxy- 2.02822.803 benzothiazol-2-yl)-5- isoxazol-5-yl-2H-pyrazol-3- ylamine4-(5-Fluoro-6-methoxy- 1.9 uM 24.5 uM 10.9 uM 5.2 uM >25 5.6 uMbenzothiazol-2-yl)-5-methyl- 1H-pyrazol-3-ylamine 4-(5-Fluoro-6-methoxy-6.1 uM 17.5 uM 14.7 uM 23.4 uM >25 10.9 uM benzothiazol-2-yl)-5-piperazin-1-yl-2H-pyrazol-3- ylamine 4-(5-Fluoro-6-methoxy- 1.4uM >25 >25 7.7 uM >25 >25 benzothiazol-2-yl)-5-pyridin-4-yl-2H-pyrazol-3-ylamine 4-(5-Fluoro-6-methoxy- 16.6 uM >25benzothiazol-2-yl)-N342- (3H-imidazol-4-yl)-ethyly1H-pyrazole-3,5-diamine 4-(5-Fluoro-6-methyl- >25 >25benzothiazol-2-yl)-2H- pyrazol-3-ylamine 4-(5-Fluoro-6-methyl- 7.822 >25benzothiazol-2-yl)-5-methyl- 2H-pyrazol-3-ylamine4-(5-Fluoro-benzothiazol-2- 10.3 uM 22.6 uM 10.7 uM 5.0 uM 20.7 uM 18.5uM yl)-5-methyl-2H-pyrazol-3- ylamine 4-(6-Chlorobenzothiazol-2- 5.6uM >25 >25 7.6 uM 16.7 uM 7.3 uM yl)-5-methyl-2H-pyrazol-3- ylamine4-(6-Dimethylaminomethyl- 2.351 >25 5-fluoro-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3- ylamine 4-(6-Dimethylaminomethyl- 1.5 uM 19.1 uMbenzothiazol-2-yl)-5-methyl- 2H-pyrazol-3-ylamine4-(6-Fluoro-benzothiazol-2- 11.6 uM N >25 13.9 uM >25 ~25yl)-1H-pyrazol-3-ylamine 4-(6-Methoxy-benzothiazol- 2.8 uM >25 18.5 10.121.1 24.5 2-yl)-5-methyl-1H-pyrazol-3- ylamine4-(6-Methoxy-benzothiazol- 5.9 uM 20.5 uM 2-yl)-5-piperazin-1-yl-2H-pyrazol-3-ylamine 4-Benzothiazol-2-yl-1H- 12.6 uM N 7.6pyrazol-3-ylamine 4-Benzothiazol-2-yl-5-(3- 13 uM ~25dimethylamino-propyl)-2H- pyrazol-3-ylamine 4-Benzothiazol-2-yl-5-(3-9.1 uM 24.6 uM 14.8 uM methylamino-propyl)-2H- pyrazol-3-ylamine4-Benzothiazol-2-yl-5-(4- 16.4 uM 23.6 uM dimethylamino-butyl)-2H-pyrazol-3-ylamine 4-Benzothiazol-2-yl-5-(4- 11.3 uM ~25methylamino-butyl)-2H- pyrazol-3-ylamine 4-Benzothiazol-2-yl-5- 7.9 uM22.112 uM >25 16.1 uM >25 16.2 uM piperazin-1-yl-2H-pyrazol-3- ylamine4-Benzothiazol-2-yl-5- 2.3 uM >25 5.1 >25 >25 18.5pyridin-4-yl-2H-pyrazol-3- ylamine 4-Benzothiazol-2-yl-N3-(3- 5.7 uM >25N 13.2 uM >25 12.8 uM dimethylamino-propyl)-1H- pyrazole-3,5-diamine5-(3-Amino-propyl)-4- 7.0 uM 22.8 uM 13.2 uM 17.3 uM 18.9 uM 10.8 uMbenzothiazol-2-yl-2H- pyrazol-3-ylamine 5-(4-Amino-phenyl)-4- 1.0 uM21.5 4.3 2.3 21.3 17.7 benzothiazol-2-yl-2H- pyrazol-3-ylamine5-Methyl-4-(4,5,6-trifluoro- 18.3 uM >25 benzothiazol-2-yl)-1H-pyrazol-3-ylamine 5-Methyl-4-(6- .5 uM .9 uM methylaminomethyl-benzothiazol-2-yl)-2H- pyrazol-3-ylamine 5-Methyl-4-(6-morpholin-4- 3.8uM >25 ylmethyl-benzothiazol-2-yl)- 2H-pyrazol-3-ylamineN-[2-(5-Amino-3-methyl-1H- 9.9 uM >25 18.442 uM 7.7 uM 18.4 uM 12.6 uMpyrazol-4-yl)-benzothiazol- 6-yl]-acetamide N₃-(2-Amino-ethyl)-4- 16.6uM >25 23.9 Um N >25 >25 benzothiazol-2-yl-1H- pyrazole-3,5-diamineN3-(2-Dimethylamino- 11.0 uM >25 17.147 uM >25 >25 >25ethyl)-4-(5-fluoro-6- methoxy-benzothiazol-2-yl)-1H-pyrazole-3,5-diamine N3-(3-Dimethylamino- 10.2 uM ~25propyl)-4-(5-fluoro-6- methoxy-benzothiazol-2-yl)-1H-pyrazole-3,5-diamine

7. Inhibition of Nitric Oxide

Peritoneal exudate macrophages were isolated by peritoneal lavage withice-cold sterile physiological saline 24 hours after intraperitonealinjection of BALB/c mice with 0.3 ml of sterile zymosan A (1 mg/0.5 mL0.9% saline). Cells were washed, resuspended in RPMI 1640 supplementedwith 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin,and 5% FBS. 1.5×10⁵ cells/well were seeded in 96-well plates andfollowed by 3 hour incubation at 37° C. with 5% CO₂ (macrophages wereallowed to attach) cells were stimulated with LPS (0.5 mg/mL) and IFN-γ(100 U/mL) in the absence or presence of the test compounds. Alltreatments were replicated six times. Cells were incubated for anadditional 24 hours, and cell free culture supernatants from each wellwere collected for NO and cytokine determination. The remaining cellswere stained with crystal violet to determine effect of the compound oncell survival.

For discussion of the stimulation of primary mouse peritonealmacrophages for NO and cytokine determination see, e.g., Calandra T.,Spiegel L. A., Metz C. N., and Bucala R. Proc Natl Acad Sci USA (1998)95(19): 11383-8; Lu L., Bonham C. A., Chambers F. G., et al. J. Immunol.(1996) 157(8): 3577-86; Keil D. E., Luebke R. W., and Pruett S. B. Int JImmunopharmaco” (1995) 17(3): 157-66; and Skeen M. J., Miller M. A.,Shinnick T. M., et al. J. Immunol. (1996) 156(3): 1196-206.

In testing of representative compounds, the release of IFNg fromsplenocytes was inhibited compared to controls. See Table 2, supra.

Inhibition of NO Release Determination:

The production of NO was determined by assaying culture supernatants forNO₂ ⁻, a stable reaction product of NO with molecular oxygen. Briefly,100 μL of culture supernatant was reacted with an equal volume of Griessreagent at room temperature for 10 minutes. The absorbance at 550 nm wasdetermined. All measurements were performed six times. The concentrationof NO₂ ⁻ was calculated by comparison with a standard curve preparedusing NaNO₂.

For discussion of measuring nitric oxide in tissue culture supernatants,see, e.g., Amano F., and Noda T. “Improved detection of nitric oxideradical (NO) production in an activated macrophage culture with aradical scavenger, carboxy PTIO and Griess reagent” FEBS Lett. (1995)368(3): 425-8; Archer S. “Measurement of Nitric oxide in biologicalmodels” (1993) FASEB J. 7:349-360, and Amin A. R. “Regulation of nitricoxide and prostaglandin E2 production by CSAIDS (SB203580) in murinemacrophages and bovine chondrocytes stimulated with LPS” Inflamm Res.(1999) 48(6):337-43.

TABLE 3 Percent Inhibition Of Nitric Oxide On Stimulated Macrophages At25 μm for Representative Compounds Chemical Name % Inhibition2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole- 100 6-sulfonic acidamide 2-(5-Amino-4-benzothiazol-2-yl-2H-pyrazol-3-ylamino)- 78.6cyclopentanol 4-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-6-ethyl- 99.3benzene-1,3-diol 4-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-phenol 69.64-(6-Bromo-benzothiazol-2-yl)-5-methyl-1H-pyrazol- 100 3-ylamine4-(6-Fluoro-benzothiazol-2-yl)-5-methyl-1H-pyrazol- 61.5 3-ylamine4-(6-Methanesulfonyl-benzothiazol-2-yl)-5-methyl-2H- 52.9pyrazol-3-ylamine 4-(6-Methoxy-benzothiazol-2-yl)-5-methyl-1H-pyrazol-100 3-ylamine 4-Benzothiazol-2-yl-1H-pyrazole-3,5-diamine 53.64-Benzothiazol-2-yl-5-methyl-1H-pyrazol-3-ylamine 96.44-Benzothiazol-2-yl-5-methylsulfanyl-1H-pyrazol- 69.2 3-ylamine4-Benzothiazol-2-yl-5-phenyl-1H-pyrazol-3-ylamine 76.74-Benzothiazol-2-yl-5-pyrrolidin-1-yl-1H-pyrazol- 52.6 3-ylamine4-Benzothiazol-2-yl-N5-quinolin-6-yl-1H-pyrazole- 66.1 3,5-diamine

Example 43 In Vitro Angiogenesis Assay

Angiogenesis, the formation of new blood vessels from pre-existingendothelium, is a critical process involved in numerous physiologicaland pathological conditions. Disruption of this tightly regulatedprocess has been implicated in both chronic inflammation and solidtumour growth. The Matrigel™ morphogenesis assay is an in vitro modelused to mimic the process by which endothelial cells form capillaries invivo. Human umbilical vein endothelial cells (HUVECs) were plated overmatrigel, a complex mixture of solubilized basement membrane components,and cultured in serum poor medium with specific growth factors and inthe presence of the test compound. HUVEC cells cultured for 24 hours inM199 with 0.5% FCS were plated at 6×10⁵ cells/well in 12-well platespre-coated with 300 μL of Matrigel (10.7 mg/mL) in M199 with 0.5% FCS inthe presence of VEGF (1 ng/mL), and in the absence or presence of thetest compounds. After 5 hours of incubation in a 5% CO₂-humidifiedatmosphere at 37° C., the three-dimensional organization of the cells(the capillary-like structures) was examined using an invertedphotomicroscope. The cells were fixed with crystal violet (0.05% in 20%ethanol) and photographed using a digital camera. Qualitative analysiswas accomplished by comparing the pattern, size and integrity of thevessels formed in the test wells with those of the VEGF control wells.Quantitative analysis was performed on the images collected using theImage-Pro Plus software program. See Table 4 for results with selectedcompounds.

For further discussion regarding in vitro angiogenesis assay, see, e.g.,Grant D. S., Lelkes P. I., Fukuda K., and Kleinman H. K. “Intracellularmechanisms involved in basement membrane induced blood vesseldifferentiation in vitro” In Vitro Cell Dev Biol. (1991) 27A(4):327-36;Kubota Y., Kleinman H. K., Martin G. R., and Lawley T. J. “Role oflaminin and basement membrane in the morphological differentiation ofhuman endothelial cells into capillary-like structures” J. Cell Biol.(1988) 107(4):1589-98; Passaniti A., Taylor R. M., Pili R., et al. “Asimple, quantitative method for assessing angiogenesis andantiangiogenic agents using reconstituted basement membrane, heparin,and fibroblast growth factor” (1992) Lab. Invest. 67:519-528.

TABLE 4 Percent Inhibition of Angiogenesis Chemical Name % Inhibition4-Benzothiazol-2-yl-1H-pyrazole-3,5-diamine 324-Benzothiazol-2-yl-5-pyrrolidin-1-yl-1H-pyrazol- 50 3-ylamine4-Benzothiazol-2-yl-5-methylsulfanyl-1H-pyrazol- 84 3-ylamine4-Benzothiazol-2-yl-5-methyl-1H-pyrazol-3-ylamine 64

Example 44 Tumor Cell Migration Assay

Tumor cell migration assay was conducted in the similar way as describedin Example 16 except that the plates used were constructed with only aporous membrane dividing a top and bottom chamber without the additionalthin Matrigel™ layer on top of the membrane (BD Fluoroblock™ plates).The percent inhibition of migration was determined in the same way asillustrated in Example 16.

See, e.g., Crouch M. F. (2000) “An automated fluorescence based assay ofneurite formation” J Neurosci Methods 104(1):87-91; and Repesh L. A.(1989) “A new in vitro assay for quantitating tumor cell invasion”Invasion Metastasis 9(3):192-208 for additional discussion aboutinvasion and migration assays.

Representative compounds were tested and the results are shown in Table5.

TABLE 5 Percent Inhibition of Migration in PC3 Cells at 25 μM CompoundCompound Name % Inhibition4-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-6-ethyl- 80.0 benzene-1,3-diol4-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-benzene- 44.8 1,3-diol4-Benzothiazol-2-yl-1H-pyrazol-3-ylamine 26.34-Benzothiazol-2-yl-1H-pyrazole-3,5-diamine 27.94-Benzothiazol-2-yl-N5-ethyl-1H-pyrazole-3,5- 61.5 diamine4-Benzothiazol-2-yl-5-pyrrolidin-1-yl-1H-pyrazol- 47.4 3-ylamine4-Benzothiazol-2-yl-5-methylsulfanyl-1H-pyrazol- 51.6 3-ylamine4-Benzothiazol-2-yl-5-methyl-1H-pyrazol-3-ylamine 41.74-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-phenol 50.92-(4-Benzothiazol-2-yl-1H-pyrazol-3-yl)-phenol 17.14-(5-Trifluoromethyl-benzothiazol-2-yl)-1H-pyrazol- 56.1 3-ylamine4-Benzothiazol-2-yl-5-ethyl-1H-pyrazol-3-ylamine 54.24-Benzothiazol-2-yl-N5-quinolin-6-yl-1H-pyrazole- 81.0 3,5-diamine2-(5-Amino-4-benzothiazol-2-yl-2H-pyrazol-3-ylamino)- 23.1 cyclopentanol4-(6-Methoxy-benzothiazol-2-yl)-5-methyl-1H-pyrazol- 79.4 3-ylamine2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-benzothiazole- 16.7 6-sulfonic acidamide 4-(6-Fluoro-benzothiazol-2-yl)-5-methyl-1H-pyrazol- 45.3 3-ylamine2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-benzothiazole- 30.5 6-sulfonic acidmethylamide 2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro- 42.9benzothiazole-6-sulfonic acid amide-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro- 38.9benzothiazole-6-sulfonic acid (2-hydroxy-ethyl)-amide

Example 45 Irritant Contact Dermatitis Model (ICD)

Female Balb/c (H2-^(d)) mice were used in this experiment (n=8). ICD wasinduced with phorbol 12-myristate 13-acetate (PMA), 4 μg/ear (in 20 μLacetone). Dexamethasone was used as a positive control (0.5 mg/kg) andwas administered s.c. in 50 mL volume prior to irritation. The irritantwas painted onto the dorsal side of the right ear pinna. The testcompounds were delivered via oral gavage at a dose between 50-300 mg/kg(10 mL/kg). Ear thickness was measured with a spring-loaded dialmicrometer before irritation and at 3, 6 and 24 hours after painting theirritant. The efficacy of the anti-inflammatory effect of the testcompounds was determined by comparison of the thickness of the inflamedear and the control ear.

Example 46 Orthotopic Lung Model

NCI-H460 human lung large cell carcinoma cells were harvested bytrypsinization and adjusted to a final concentration of 1×10⁶ cells/80mL. Male nude rats (CR:NIH-RNU) were endobronchially implanted with1×10⁶ tumor cells using a 20 gauge, 2 inch Teflon™ catheter passed intothe right caudal lobe via a small tracheotomy incision.

Implantation of tumor fragments. These tumor-bearing rats weresacrificed at three weeks following implantation and their tumorsharvested in cold RPMI 1640. Viable tumor was cut into 1-2 mm diameterpieces by “crossed scalpels” technique. A 50 mg portion was placed intoa 16 gauge, 2 inch Teflon™ catheter and implanted into 6-week-old malenude rats using a similar technique. Animals were treated withAugmentin™ supplement at 0.35 mg/mL in water for 2 weeks.

The test compound was prepared fresh each day by dissolving it in anacceptable recipient at 10 mg/mL under sterile conditions. Cisplatininjection, 1 mg/mL, was obtained from the hospital pharmacy.

There were four arms in the study: control; test compound alone;cisplatin alone; test compound and cisplatin combination. Also, therewere two groups in the study: in group I, all animals were followeduntil death to assess maximum length of survival and in group II, allanimals were simultaneously sacrificed from each treatment arm ascontrol animals became severely cachectic or died. This allows us todirectly compare, at the same point in time, the therapeutic effects ofeach study arm on tumor related endpoints, such as primary tumor weight,tumor/body weight ratio, mediastinal lymph node weight and metastaticpattern. Renal and liver functions of each animal were also examined byserum biochemistry to assess possible toxicities.

Both test compound (5 mg daily) and cisplatin (5 mg/kg weekly for 3weeks) were administered by intraperitoneal injection. Treatmentcommenced 7 days and 14 days post implantation for the test compound andcisplatin, respectively. Animals were sacrificed when they showed signsof significant morbidity or impending death. At necropsy the heart-lungblocks, kidney, brain, and chest wall were removed, serially sectioned,stained, and examined in a blinded fashion by a pathologist.

Statistical analysis for length of survival, primary tumor, body, andmediastinal lymph node weight were evaluated using ANOVA or unpairedStudent's t-test. Incidence of metastasis was evaluated by using acontingency table with Fisher's exact test. Differences of P<0.05 wereconsidered to be significant.

Immunocytochemistry. The H-460 cell line was seeded into 8-chamberslides (10⁴ cells/well) and treated with 25 μM of the test compoundafter reaching a confluency of 60 to 80%. Cells were harvested at 2, 4,8 and 24 hours after treatment and incubated overnight at 4° C. with theprimary antibodies. For phosphorylated Akt/PKB expressionanti-phospho-Akt/PKB (Ser-473), was used at a concentration of 2 μg/mL,followed by incubation with the secondary antibody, biotinylatedrabbit-IgG at a concentration of 7 μg/mL. For phosphorylated GSK-3βexpression anti-phospho-GSK-3β (Ser-9), a concentration of 6 μg/mL wasused, followed by incubation with the same secondary antibody.Streptavidin-peroxidase was used as a detection system. DAB was used aschromogen and counterstaining was performed with hematoxylin. Slideswere assessed as either positive or negative according to the amount andintensity of staining. Phospho-Akt/PKB and phospho-GSK-3β reactivity wasquantitated by computerized image analysis using an Image-Pro™ systemand conventional light microscopy.

Example 47 ILK Expression is High in Human Psoriatic Skin as Compared toNormal Skin

The thickness of the epidermal layer within psoriatic plaques isdramatically greater than that of normal skin of healthy individuals orthe uninvolved skin of the psoriasis patient.

To test for ILK expression, skin samples were obtained from a humansubject with healthy skin and from patients suffering from theimmune-mediated condition psoriasis. Skin preparations were processedusing routine formalin-fixation and paraffin embedding techniques.Sections were cut and treated with antigen retrieval methodology andstained with a rabbit anti-ILK polyclonal antibody (catalogue #06-592,Upstate Biotechnology, Lake Placid N.Y.). Sections were then incubatedwith peroxidase-conjugated goat anti-rabbit polyclonal antibody. Slideswere then developed using standard techniques.

In normal skin, a low level of ILK expression was evident in thesupra-basal layers of skin keratinocytes. These supra-basal layers ofskin keratinocytes were almost certainly undergoing the process ofterminal differentiation. The staining intensity for ILK was moreintense for keratinocytes near the outer keratin layer. Little or no ILKstaining was observed for the dermal vascular endothelium. In contrast,staining for ILK was highly intense for the hyper-proliferativekeratinocytes within the plaques of patients with psoriasis patients.Within the dermal region of psoriatic patient plaques, cells comprisingthe vasculature stained strongly for ILK. Further, some of theinflammatory cells present within the dermal region stained positivelyfor ILK. Overall, in contrast to normal skin, ILK was expressed at muchhigher levels within the epidermal and dermal regions within skinplaques of patients with psoriasis.

Example 48 Expression of ILK in Psoriatic Tissue Correlates withSeverity of Disease

The expression of ILK within psoriatic skin was evaluated for a seriesof plaque biopsy samples obtained from a patient over a 3-month period.The presence and expression pattern of ILK was evaluated byimmunohistological analyses. All sections were stained at the same time.For psoriasis, the disease-state can be gauged by the relative thicknessof the epidermis. For the series of biopsy samples evaluated, expressionlevels of ILK closely paralleled the psoriasis disease-state at thetissue level.

The first sample (panel A), was obtained at screening while the patientwas experiencing active disease. Staining for ILK was intense for thekeratinocytes within the target plaque. Within the dermal region of theplaque, cells within the vasculature as well as cells that hadinfiltrated the region also stained strongly for ILK. The second sample(panel B) was obtained one month later, a time when disease activity hadfurther intensified. ILK staining intensity with this sample was muchstronger than for the first sample. The third sample was takenapproximately 4 weeks after sample B, a time during which this subjectwas exhibiting an improvement in his disease and a reduction inepidermal thickness. For this sample (panel C) there was a correspondentreduction in ILK staining intensity, both for the epidermalkeratinocytes and within cells of the dermal vasculature. Sample 4 wasobtained 3 months after sample 1, at a time when the subject wasexperiencing a flare in disease activity. Epidermal thickness for sample4 was greater than that of sample 3. At this time, an increase in ILKstaining intensity was evident within the dermal vasculature andcellular infiltrate as well as for the epidermal keratinocytes (panelD). Thus, expression levels of ILK within the psoriatic plaque variedwith disease activity with high ILK expression correlating with symptomsof active disease.

Example 49 Anti-ILK Compound Inhibits Influx of Neutrophils into Site ofInflammation

Administration of certain pro-inflammatory agents, such as zymosan, intothe peritoneal cavity of mice elicits a rapid influx of neutrophils intothis region. The migration of these cells into the peritoneal cavityrequires the coordinate interaction of cytokines, chemokines and celladhesion molecules. Such a system can be used to evaluate the action ofcompounds with potential for modifying the migration of cells inresponse to pro-inflammatory stimuli.

When zymosan was administered to mice, peritoneal cavity neutrophilnumbers increased by approximately 4-fold within 4 hours. However, if acompound of the invention was given orally at 200 mg/kg at the time ofzymosan administration, cells numbers within the peritoneal cavity wereequivalent to those of animals that received a saline control solvent 4hours before. Thus, a compound with specific in vitro anti-ILK activitycan affect the influx of cells into a tissue following the delivery of astrong pro-inflammatory signal in vivo.

For a discussion of acute inflammation models, including Irritantcontact dermatitis (ICD) and allergic contact dermatitis (ACD), see,e.g., Artik S., von Vultee C., Gleichmann E., Schwarz T., and Griem P.“Nickel allergy in mice: enhanced sensitization capacity of nickel athigher oxidation states” J. Immunol. (1999) 163(3):1143-52; Becker D.,Lempertz U., Enk A., Saloga J., and Knop J. “Contact sensitizersmodulate mechanisms of receptor-mediated endocytosis but not fluid-phaseendocytosis in murine epidermal Langerhans cells” Exp. Dermatol. (1995)4(4 Pt 1):211-7; Griswold D. E., Martin L. D., Badger A. M., Breton J.,and Chabot-Fletcher M. “Evaluation of the cutaneous anti-inflammatoryactivity of azaspiranes” Inflamm. Res. (1998) 47(2):56-61; and Moreno J.J. “Effect of retinoids on dermal inflammation and on arachidonic acidmobilization and metabolism in murine 3T6 fibroblasts retinoids,arachidonate release and metabolism” Int. J. Immunopharmacol. (1996)18(8-9):459-65.

Example 50 Demonstration of Ilk Inhibition as Therapeutic Interventionin Renal Disorders In Vitro Murine Podocyte Model

1. Survival Assay in Podocytes

The treatment of glomerular visceral epithelial cells (podocytes) withhigh concentrations of puromycin aminonucleoside (PAN) causessignificant cytotoxic effects. An assessment of cell viabilitysubsequent to a cytotoxic exposure can be judged qualitatively byexamining changes in cell phenotype after staining with crystal violetor determined quantitatively by employing a cytotoxic assay using thesoluble tetrazolium salt[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] (MTS). Such a system can be used to evaluate the survivaleffects that ILK compounds have on podocytes following exposure to PANor other cytotoxic agents.

The cell line employed for the podocyte model was the murine K5P5. Cellswere maintained and grown in tissue culture flasks (T75) in RPMI1640+10% FBS medium supplemented with 10 U/ml of IFNγ and incubated at33° C., 5% CO₂. The cells should be propagate to a maximum of 20passages.

Cells were trypsinized (0.25%), collected from the tissue culture flaskscentrifuged, resuspended and counted.

Collagen coated flasks were prepared by adding 1 ml/25 cm² of a 100μg/ml Collagen I (Biochrom) and allowing to bind for 1 hour at 37° C.The flasks were then washed twice with PBS to remove any unboundCollagen I.

Podocyte cellular differentiation was initiated by seeding the cells(1×10⁶) on Collagen I coated flasks (T150) in RPMI 1640+10% FBS mediumwithout IFNγ. The flasks were incubated at 37° C., 5% CO₂ for 3 days.

Cells were trypsinized (0.25%), collected from the tissue culture flaskscentrifuged, resuspended and counted.

Podocyte cellular differentiation was continued by seeding the cells(7×10⁵) on Collagen I coated flasks (T150) in RPMI 1640+10% FBS mediumwithout IFNγ. The flasks were incubated at 37° C., 5% CO₂ for 4 days.

Collagen coated 96-well plates were prepared by adding 75 μl/well of a100 μg/ml Collagen I (Biochrom) and allowing to bind for 1 hour at 37°C. The flasks were then washed twice with PBS to remove any unboundCollagen I.

Cells were trypsinized (0.25%), collected from the tissue culture flaskscentrifuged, resuspended and counted. Podocyte cellular differentiationwas continued by seeding the cells (3.5×10³) on Collagen I coated96-well plates in 10% FBS RPMI 1640 medium without IFNγ. The flasks wereincubated at 37° C., 5% CO₂ for 3 days.

On day 10, the supernatant was removed from the 96-well plates andreplaced with 2% FBS RPMI 1640 overnight.

For test compounds, cell culture media (e.g., RPMI 1640+2% FBS), 10×compound solution of final desired concentration from 40 mM stockcompounds was prepared.

10 μl of this 10× compound solution is added to the 80 μl of cellsalready present in the 96-well plates. For cell undergoing cytotoxictreatment, puromycin aminonuceloside (PAN, Sigma P7130) was added at a10× concentration to 90 μl of cells. The positive control is PANtreatment without compound. The negative control is 100% DMSO diluted tothe same factor as the compounds without PAN.

The plates are incubated at 37° C., 5% CO₂ for 48 to 72 hours dependingon the PAN concentration (i.e. lower concentrations of PAN required alonger incubation period). The medium is aspirated after plates are spunat 2400 rpm for 10 min at ambient temperature. 100 μl of 1×DPBS (withoutCaCl₂, without MgCl₂) is added to each well.

The MTS (Promega) is prepared under low lighting conditions bydissolving 4.0 g of MTS in 1.8 I of 1×DPBS. The solution is allowed tosit for 10 minutes and pH adjusted to 6.2. H₂O is added to 2 L. 100 mlof PMS is added (dissolve 0.92 g of PMS in 1.01 of 1×DPBS) and 20 μl ofMTS is added slowly to each well and incubated for 4 hours at 37° C.Absorbance is measured at 490 nm wavelength with a microplate reader.

A kit available to do this assay is “CellTiter 96® AQ_(ueous) Kit(G5421)” by Promega.

The values for four replicates are averaged and % inhibition iscalculated at follows:% survival=100−[(AVG treatment−AVG positive control)/(AVG negativecontrol−AVG positive control)*100].

The cyto-protective activity of 5 representative compounds is shownTable 6. Several of the IC₅₀ measurements were in the range of 2.5 to12.5 μM with two of the compounds reaching 80% survival of K5P5podocytes in the presence of PAN (data not shown). To further evaluatethe compound effects that were observed with the MTS assay, cells wereanalyzed for changes in morphology. The cells were photographed afterbeing stained with crystal violet. The results demonstrated that theincrease in cell survival observed by the MTS assay correlates with therestoration of the normal phenotype of the podocytes. Hence, increaseddoses of compound resulted in an increase in cell size, cell spreading(adhesion) and overall cell number (data not shown).

TABLE 6 % Survival on PAN a treated K5P5 podocyte cell line (IC₅₀, μM)Chemical Name % Survival 2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-benzo-12.5 thiazole-6-sulfonic acid methylamide2-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro- 30%benzothiazole-6-sulfonic acid amide @ 12.52-(5-Amino-3-methyl-1H-pyrazol-4-yl)-5-fluoro- 30%benzothiazole-6-sulfonic acid (2-hydroxy-ethyl)-amide @ 25  4-(5-Fluoro-6-methoxy-benzothiazol-2-yl)-5-furan-  2.52-yl-2H-pyrazol-3-ylamine 4-(6-Dimethylaminomethyl-benzothiazol-2-yl)-5-12.5 methyl-2H-pyrazol-3-ylamine

2. Transgenic Mouse Model

Mice transgenic for the bovine growth hormone (GH) under amethallothionein I promoter are used (Wanke, R., et al. PediatricNephrol (1991) 5:513-521). Genotype can be confirmed by genomic PCR withbovine GH specific primers (Wanke, R., et al supra). Glomeruli can beisolated after pooling kidneys from two or more animals. For the animalmodel, accelerated nephrotoxic serum nephritis (NTX) is induced in 4 to6 week old females as previously reported (Schadde, E., et al. NephrolDial Transplant (2000) 15:1046-1053; Neugarten, J., et al. J Am SocNephrol (1995) 5:1903-1909). Five days after preimmunization with rabbitIgG, 400 μg of a protein A purified IgG fraction of a nephrotoxic rabbitanti-murine GBM antiserum is intravenously injected, while controlsreceive carrier only. Mice in each group are sacrificed after 0, 2, and7 days and a pooled glomerular fraction is obtained from each group forexpression analysis. Albuminuria is determined using a commerciallyavailable mouse albumin specific ELISA system (Exocell, Philadelphia,PE).

3. Measuring Levels of ILK from Podocytes

To assess the efficacy of a candidate pyrzolylbenzothiazole compound invivo, the following podocyte extraction method may be used. Single cellRT-PCR is performed as described in Schroppel, B., et al. Kidney Int(1998) 53:119-124. Freshly dissected glomeruli from CD-1 mice aretransferred to a patch clamp apparatus. Single podocytes are selectivelyharvested by aspiration of the cells into a micropipette. Reversetranscribed and RT-PCR is performed essentially as described above, butusing 50 instead of 30 cycles. Perfusion medium aspirated next to aglomerulus is processed in parallel and serves as negative control.Single cell ILK RT-PCR product identity is verified by restrictiondigest. Single podocyte RNA is quantified using published real-timeRT-PCR technology (Heid, C. A., et al. Genome Res (1996) 6:986-994). Fordetermination of ILK copy number per single podocyte cDNA, a standardcurve of serial dilutions of ILK plasmid cDNA with known copy numbers isemployed. ILK copies per podocyte cDNA are calculated using the Ct valueminus the dilution factor and the standard curve (y=−1.6227 Ln(x)+39with R2=0.9935) generated from duplicate amplification reactions of logfold dilutions between 100,000 and 10 ILK plasmid copies.

4. Adriamycin-Induced Proteinuria Model

This model, which results in focal glomerular sclerosis (FGS), is welldescribed in Wang, Y., et al. Kidney Int (2000) 58:1797-1804. Groups ofBALB/c mice are injected intravenously on day 0 with a single dose ofAdriamycin (ADR, doxorubicin hydrochloride, Pharmacia & Upjohn) at 10-11mg/kg, or vehicle control. Six to eight animals in each group areanalyzed.

GROUP TREATMENT Negative Intravenous carrier on day 0, vehicle dailycontrol group from day 0 Positive Intravenous ADR on day 0, vehicledaily control group from day 0 Test group Intravenous ADR on day 0,various doses of ILK inhibitor from day 0

Pyrazolylbenzothiazole compounds are administered orally,intraperitoneally or by subcutaneous infusion pump, in daily dosesranging from 0.01-200 mg/kg, beginning on day 0. Vehicle (carrier)controls are administered in equivalent volumes by the same routes.

Experimental readouts include weekly body weight, urine volume, urinaryprotein, serum creatinine and albumin, and terminal histopathology.Negative control mice demonstrate no significant changes in experimentalreadouts. The positive control group demonstrates significant changesassociated with rapid progressive renal disease (FGS) using experimentalreadouts, namely proteinuria, hypoalbuminemia, hypercreatininemia, andprogressive renal injury by histology. In the experimental groupstreated with various doses of pyrazolylbenzothiazole compound, decreasesin measured parameters of progressive renal disease are demonstratedcompared to the positive control group, indicating that administrationof pyrazolylbenzothiazole compounds results in therapeutic benefit inthis model of acute progressive focal glomerular sclerosis.

5. Murine Unilateral Ureteral Obstruction Model

This model results in epithelial-mesenchymal transdifferentiation inrenal fibrosis and is described in Vielhauer V., et al. J Am Sox Nephrol(2001) 12: 1173-1187. Briefly, female inbred C57BL/6 mice weighing ca.20-26 g are obtained and kept under a ca. 12-h light/dark cycle. Foodand water are available ad libitum. Under general anesthesia, unilateralureteral ligation resulting in UUO is performed by ligating the leftdistal ureter with a 2/0 Mersilene™ suture through a low midlineabdominal incision. Unobstructed contralateral kidneys serve ascontrols.

Experimental Plan for UUO Model

GROUP (8-10 mice) PRE-TREATMENT TREATMENT Negative Sham operated miceReceive carrier only for control group 10 days Positive Mice with oneReceive carrier only for control group obstructed kidney 10 days Testgroup Mice with one Receive various doses of obstructed kidney ILKinhibitor for 10 days

Test compounds are administered orally, intraperitoneally or bysubcutaneous infusion pump, in daily doses ranging from 0.01-200 mg/kg.Vehicle (carrier) controls are administered in equivalent volumes by thesame routes. Experimental readouts included histological fibrosisscores, serum urea, collagen levels and ILK mRNA expression. Analysis ofILK mRNA levels are also performed in infiltrating cells (macrophagesand T-cells) after cell sorting in renal fibrosis in the UUO model.Negative control (sham operated) mice demonstrate no significant changesin experimental readouts. The UUO control group demonstrates significantchanges associated with renal fibrosis in the ligated kidney usingexperimental readouts. Also observed in these animals is an increase inILK mRNA induction. In the experimental groups treated with variousdoses of pyrazolylbenzothiazole compound, the non-ligated kidneys areused as internal controls, and the non-ligated kidneys demonstrate nosignificant changes associated with renal tubulo-interstitial fibrosisusing experimental readouts, however the damaged kidneys demonstratedecreases in measured parameters of renal fibrosis compared to the UUOcontrol group. This result indicates that administration ofpyrazolylbenzothiazole compounds results in therapeutic benefit in thismodel of renal tubulo-interstitial fibrosis.

Example 51 Treatment of AMD Using an Ilk Inhibitor as an Adjunct toVisudyne™ Therapy

Therapeutic effect of a pyrazolylbenzothiazole compound in AMD isevaluated using visual acuity as the primary clinical outcome. Patientswith subforveal CNV lesions caused by AMD are examined for the presenceof lesions that meet the inclusion criteria. The inclusion criteria aredefined as the presence of lesions measuring 5400 μm or less in greatestlinear dimension with evidence of classic CNV and best-corrected visualacuity of approximately 20/40 to 20/200 based on fluoresceinangiographic and visual acuity examination. Those determined asqualified for the treatment of AMD are randomly assigned to 4 groups.Group A, B, and C are treated with standard Visudyne™ therapy with anadjunct therapy using an ILK inhibitor. Patients of Group D are treatedwith standard Visudyne™ therapy in combination with a placebo of the ILKinhibitor.

For standard Visudyne™ therapy, patients are administered with 30 ml ofVisudyne™ (0.15 mg per kilogram of body weight). The administration isby intravenous infusion over a period of 10 minutes. Fifteen minutesafter the end of the infusion, the laser light is applied for 83 secondsto the CNV lesion through a fundus contact lens of known magnificationto result in a light exposure of 50 J/cm². A circular spot ofapproximately 6000 microns encompassing the area of the lesion isexposed to the laser light.

For the adjunct therapy, patients of groups A, B, and C receive a dailyoral administration of an ILK inhibitor at the dose of 5, 10, 20 mg perkilogram body weight, respectively. The adjunct treatment commencesthree days after the patient receiving the standard Visudyne™ therapyand continues for a period of one month.

As follow-up, patients are examined every three months. At eachregularly scheduled follow-up visit, best-corrected visual acuitymeasurement, contrast threshold measurement, ophthalmoscopicexamination, stereoscopic fundus photography, and fluoresceinangiography are performed.

Example 52 Treatment of Diabetic Retinopathy Using an ILK Inhibitor

Therapeutic effect of a pyrazolylbenzothiazole compound in proliferativediabetic retinopathy is evaluated using visual acuity as the primaryclinical outcome. Patients with proliferative diabetic retinopathy andvisual acuity of 20/100 or better in each eye are included in theclinical evaluation. Patients are randomly assigned to 3 treatmentgroups and 1 placebo group. Group A, B, and C are treated with dailyoral administration of a pyrazolylbenzothiazole compound at the dose of5, 10, 20 mg per kilogram body weight. Patients of Group D receiveplacebo. The treatment covers a period of 24 months.

As follow-up, patents are examined every 4 months. At each regularlyscheduled follow-up visit, best-corrected visual acuity measurement,contrast threshold measurement, indirect ophthalmoscopic examination,stereoscopic fundus photography, fluorescein angiography, and slit-lampexamination using 78- or 90-diopter lens are performed.

Example 53 Evaluation of ILK Expression in Ocular Vascular Tissue

This example indicates the relevance of ILK as a therapeutic target fordiseases with underling pathology of ocular neovascularization.

Post mortem baboon eye samples were subjected to immunohistologicalanalysis for the expression of ILK in ocular vasculature. Freshlyobtained tissues were snap-frozen by immersing into a Dewar container ofliquid nitrogen. Cross sections of 5-10 microns were prepared and fixedin cold acetone (−20 C). Immunohistology was performed using a rabbitanti-ILK antibody (Upstate Biotechnology Institute, NY. Cat.#06-550) andZymed Histostatin™ Plus kit (Zymed, Cat.#85-9743).

Abundant expression of ILK was detected in choroidal and retinalendothelium in post mortem baboon eye samples. Under similar condition,no significant level of ILK expression was detected in retinal pigmentedepithelial cells. In addition, no significant expression of ILK inneurons and photoreceptors was observed.

Example 54 Treatment of Corneal Neovascularization with an ILK Inhibitor

The following model provides a quantifiable in vivo assay that can beused to evaluate anti-angiogenic activity of an pyrazolylbenzothiazolecompound. Corneal neovascularization is induced by a procedure known assilver nitrate cauterization. The procedure involves topicalapplications of silver nitrate onto the cornea by gently touchingconjunctiva/limbus for one second followed by touching the centralcornea of an anesthetized mouse for 8 seconds with a silver nitrateapplicator (Graham-Field, NY, Item #1590, 75% silver nitrate, 25%potassium nitrate). Immediately after, the eye is rinsed with 10 ml ofsaline followed by topical application of Gentak™ Ophthalmic Ointment(0.3%, Gentamicin sulfate) on the eye to prevent bacterial infections.

Corneal neovascularization is recorded and evaluated by examining andphotographing the cornea daily using a stereo dissecting microscopeconnected to a color video camera and a computer. Angiogenesis isevaluated based on new blood vessel growth within previous avascularcornea using a scoring system (score of 0-4) that rates from noneovascularization to very severe neovascularization in cornea. Inaddition, upon completion of the experiment (day 5-7), cornealneovascularization is quantified using computer-assisted image analysis(Image Pro Plus, Media Cybernetics, ML) of dye-stained blood vessels inpost mortem whole corneal mounts. Corneal vasculature is stained by IVinjection of high molecular weight FITC-dextran into anesthetized micebefore euthanasia.

Animals receive daily intra-peritoneal administration of apyrazolylbenzothiazole compound at the dose of 5, 25 or 50 mg/kgcommencing on day-2 after the silver nitrate cauterization procedureuntil 24 h before the ending of the experiment. Cornealneovascularization of ILK inhibitor-treated animals is compared withthat of vehicle-treated animals.

Example 55 Treatment of Choroidal Neovascularization with an ILKInhibitor Using a Monkey Model of CNV

The following model provides an in vivo assay that can be used toevaluate therapeutic potential of pyrazolylbenzothiazole compounds forthe treatment of CNV. CNV is induced by argon green laser burns that areplaced in the maculae of cynomolgus monkeys using a modification ofRyan's model. The laser burn with size of 50 μm in diameter is inducedby exposure to 350-450 mW laser light at 514 nm for 0.1 second using anargon laser (Coherent Argon Dye Laser #920, Coherent Medical Laser, PoloAlto, Calif.).

CNV is monitored by weekly examination with fundus photography andfluorescein angiography. At the termination of the experiment (2-3months after the induction of CNV), eyes are enucleated under deepanesthesia and fixed in modified Kanovsky fixative. Bisection isperformed 20 min after fixation. Tissues are then embedded and sectionsare generated for histological and immunohistological analysis usingantibodies against vasculature-specific markers including CD-31 andVE-Cadherin. The extent of neovascularization is quantified using acomputer-assisted image analysis system with Image Pro Plus (MediaCybernetics, ML).

Animals receive daily oral administration of a pyrazolylbenzothiazolecompound at the dose of 10, 50 or 100 mg/kg for commencing after theonset of CNV (2-3 weeks after the laser treatment). As control, a groupof monkeys receive daily oral treatment with vehicle only. CNV in ILKinhibitor-treated animals is compared with that of vehicle-treatedanimals for angiographic and immunohistological evidence of CNV.

Example 56 Treatment of Retinal Neovascularization with an ILK InhibitorUsing a Mouse Model of Ischemia-Induced Retinopathy

The following model provides an in vivo assay that can be used toevaluate therapeutic potential of pyrazolylbenzothiazole compounds forthe treatment of retinopathy. This is a mouse model of retinopathy ofprematurity. Retinopathy in mice is induced in neonatal mice. Mice areexposed with their nursing dams to 75% oxygen/25% nitrogen frompostnatal day 7 to day 12, then put back to room air.

At day 17, all pups are weighed, euthanised, and perfused with 1 mlfixative (4% paraformaldhyde/8% sucrose/sodium phosphate buffer, pH 7.2)through the left ventricle of heart. Eyes are enucleated and placed infixative. The fixed tissues are paraffin-embedded and 4-μm sections arecut. Immunohistology procedure is performed to evaluate extent ofretinal neovascularization using antibodies against endothelium-specificmarkers including CD-31 and VE-cadherin. The vascular specific stainingis quantified using the computer-assisted image analysis method (ImagePro Plus, Media Cybernetics, ML).

The pyrazolylbenzothiazole compound at the dose of 5, 25 or 50 mg/kg isadministered daily through intra-peritoneal injection from day 12through day 16. The control group receives daily injection of vehicle.The inhibitory effect of the ILK inhibitor on retinal neovascularizationis determined by comparing the extent of vascular staining in micetreated with the compound and those treated with vehicle only.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A topical composition comprising apharmaceutically acceptable excipient and2-(3-Amino-5-methyl-1H-pyrazol-4-yl)-5-fluoro-benzothiazole-6-sulfonicacid methylamide.
 2. A method for relieving or causing regression ofpsoriasis or irritant contact dermatitis in a patient in need thereof,comprising topically applying a therapeutically effective amount of thetopical composition of claim
 1. 3. A method for relieving or causingregression of of psoriasis in a patient comprising topically applying atherapeutically effective amount of the topical composition of claim 1.4. A method for relieving or causing regression of irritant contactdermatitis or allergic contact dermatitis in a patient comprisingtopically applying a therapeutically effective amount of the topicalcomposition of claim 1.